Factors affecting short-term and long-term stabilities of proteins.

Proteins are marginally stable and, hence, are readily denatured by various stresses encountered in solution, or in the frozen or dried states. Various additives are known to minimize damage and enhance the stability of proteins. This review discusses the current knowledge of the mechanisms by which these additives stabilize proteins against acute stresses, and also the various factors to be considered for long-term storage of proteins in solution.

Biopharmaceutical powders: particle formation and formulation considerations.

It is well known that protein/peptide-based drug formulations are more stable in the solid state than in the liquid state, thereby offering stability advantages in ambient temperature storage, product shipping/distribution, and long-term shelf life. Novel powder-based drug delivery systems recently emerging for applications in sustained release, inhalation, intradermal delivery, etc, add more value to protein solid dosage forms. Despite great research interests in understanding the drying effects on protein stability and a large collection of publications focusing on this area, systematic accounts of powder formation techniques are lacking. This review is to summarize a number of methods currently available for protein powder preparation. Some are common methods such as lyophilization, spray drying, pulverization, and precipitation, and some methods are more recently developed such as supercritical fluid precipitation, spray-freeze drying, fluidized-bed spray coating and emulsion precipitation. In addition to examining the individual process effect on protein stability that is always the focus of formulation scientists, this review also likes to evaluate each method from a more practical sense in terms of process versatility and scalability. The conclusion is that each method has its own advantages and the use of a method is formulation and application specific. With the understanding of the principles and advantages of these methods, it can benefit our choice on selecting appropriate techniques for preparing a desired protein powder formulation for specific applications.

Activity-stability considerations of trypsinogen during spray drying: effects of sucrose.

The preparation and processing of protein pharmaceuticals into powders may impose significant stresses that could perturb and ultimately denature them. In many cases their stabilization through added excipients is necessary to yield native and active proteins. In this study, the effect of spray drying on the structure and activity of a model protein (trypsinogen) was investigated. In the absence of excipients, spray drying resulted in small losses of its enzymatic activity. Protein conformational rearrangements in the solid state (observed via FTIR) and irreversible aggregation (upon reconstitution) constituted the major degradation pathways. The irreversible unfolding in the solid state was also confirmed by solution calorimetric studies that indicated a decreased thermal stability of the spray-dried protein after reconstitution. The presence of sucrose, a thermal and dehydration stress stabilizer, induced a concentration-dependent protective effect. Protein protection was afforded even at low carbohydrate concentrations, while at specific mass ratios (sucrose-to-protein = 1:1) complete activity preservation was achieved. However, at the high end of sucrose concentrations, a small destabilization was evident, indicating that excluded volume effects may be undesirable during preparation of protein microparticles via spray drying. The profile of both the protein conformational changes and thermal stability in the solid state closely followed that of the incurred activity losses, indicating that protein stabilization during dehydration is crucial during processing of these polypeptides.

Moisture effects on protein-excipient interactions in spray-dried powders. Nature of destabilizing effects of sucrose.

The preparation of stable solid protein formulations presents significant challenges. Ultimately, the interactions between incorporated excipients and the pharmaceutical protein determine the formulation stability. In this study, moisture was utilized to probe the interactions between a model protein, trypsinogen, and sucrose in the solid state, following spray drying. Through investigation of the physical properties of the spray-dried formulations, we attempted to elucidate the mechanisms underlying the previously observed stabilizing and destabilizing effects of the carbohydrate during spray drying. Both dynamic and equilibrium moisture uptake studies indicated the presence of an optimal protein-sugar hydrogen bonding network. At low sucrose contents, a preferential protein-sucrose hydrogen bonding interaction was dominant, resulting in protein stabilization. However, at high carbohydrate concentrations, preferential sugar-sugar interactions prevailed, resulting in a phase separation within the formulation matrix. The preferential incorporation of the sucrose molecules in a sugar-rich phase reduced the actual amount of the carbohydrate available to interact with the protein and thereby decreased the number of effective protein-sucrose contacts. As a consequence, the protein could not be effectively protected during spray drying. We hypothesize that the observed phase separation at this sucrose concentration regime originates from its exclusion from the protein in solution before spray drying, further accompanied by preferential clustering of the sucrose molecules.

Precipitation of proteins in supercritical carbon dioxide.

Supercritical CO2 was used as an antisolvent to form protein particles that exhibited minimal loss of activity upon reconstitution. Organic protein solutions were sprayed under a variety of operating conditions into the supercritical fluid, causing precipitation of dry, microparticulate (1-5 microns) protein powders. Three proteins were studied: trypsin, lysozyme, and insulin. Amide I band Raman spectra were used to estimate the alpha-helix and beta-sheet structural contents of native and precipitate powders of each protein. Analysis of the Raman spectral revealed minimal (lysozyme), intermediate (trypsin), and appreciable (insulin) changes in secondary structure with respect to the commercial starting materials. The perturbations in secondary structure suggest that the most significant event during supercritical fluid-induced precipitation involved the formation of beta-sheet structures with concomitant decreases of alpha-helix. Amide I band Raman and Fourier-transform infrared (FTIR) spectra indicate that higher operating temperatures and pressures lead to more extensive beta-sheet-mediated intermolecular interactions in the precipitates. Raman and FTIR spectra of redissolved precipitates are similar to those of aqueous commercial proteins, indicating that conformational changes were reversible upon reconstitution. These results suggest that protein precipitation in supercritical fluids can be used to form particles suitable for controlled release, direct aerosol delivery to the lungs, and long-term storage at ambient conditions.

A new strategy for enhancing the stability of lyophilized protein: the effect of the reconstitution medium on keratinocyte growth factor.

PURPOSE: Protein stabilization during lyophilization has previously focused on optimization of the formulation as well as the freezing and dehydration process parameters. However, the effect of the reconstitution medium has been largely neglected. We have investigated its effect on aggregate formation using recombinant keratinocyte growth factor (KGF). METHODS: The protein was lyophilized under suboptimal conditions to induce aggregation and precipitation upon reconstitution with water. A series of additives were examined by UV spectrophotometry and size exclusion chromatography (SEC-HPLC) for their effects on decreasing the degree of KGF aggregation and precipitation by the increase in recovery of soluble monomer. RESULTS: Several additives resulted in a significant reduction of aggregation, including sulfated polysaccharides, surfactants, polyphosphates, and amino acids. A similar effect was achieved by adjusting the ionic strength of the reconstitution medium. SEC-HPLC indicated that the amount of soluble monomer was also increased by these additives suggesting that the recovery of the soluble protein correlates with the native, monomeric protein. CONCLUSIONS: These results suggest that optimization of reconstitution conditions will be a useful methodology for increasing the recovery of soluble, active proteins and that for KGF, the recovery of the soluble protein correlates with the native, monomeric form.

Optimization of lyophilization conditions for recombinant human interleukin-2 by dried-state conformational analysis using Fourier-transform infrared spectroscopy.

PURPOSE: Examination of the dried-state conformation of interleukin-2 (IL-2) was used to determine the pH conditions and stabilizers that provide optimal storage stability for the lyophilized product. METHODS: Fourier-transform infrared spectroscopy and accelerated stability studies which examined solubility, aggregate formation, and covalent cross-linking were used. RESULTS: Varying the pH in the absence of excipients resulted in dramatic differences in the dried state conformation of IL-2. At pH 7, IL-2 unfolds extensively upon lyophilization while at pH below 5 it remains essentially native. Additional unfolding was observed upon incubation at elevated temperatures. A strong direct correlation between the retention of the native (aqueous) structure during freeze-drying and enhanced stability is demonstrated. IL-2 prepared at pH 5 is approximately an order of magnitude more stable than at pH 7 with regard to formation of soluble and insoluble aggregates. A similar pH profile was observed in the presence of excipients, although the excipients alter the overall stability profile. Additional accelerated stability studies examined the stabilizers necessary for optimal stability. CONCLUSIONS: Excipients with the capacity to substitute for water upon dehydration better preserve the native structure resulting in enhanced stability. Those that have high glass transition temperatures provide the highest level of stability during storage, although they do not prevent dehydration induced unfolding.

The importance of Arg40 and 45 in the mitogenic activity and structural stability of basic fibroblast growth factor: effects of acidic amino acid substitutions.

High-affinity binding of basic fibroblast growth factor (bFGF) to the tyrosine kinase receptor requires cell-surface heparan sulfate proteoglycan or exogenous addition of heparin. The crystal structure of bFGF shows Arg40 and 45 on the surface opposite to the heparin-binding region, suggesting that these charged residues may be involved in the receptor binding. Therefore, these amino acids were mutated to aspartic acid separately or simultaneously, and also a simultaneous mutation to glutamic acid was introduced. These mutants displayed a mitogenic activity decreased greater than tenfold compared to the wild-type protein. Addition of heparin had no effect on the activity, while these mutants showed heparin-binding characteristics resembling those of the native sequence protein. The mutants exhibited decreased stability compared to the native sequence protein. Gradual changes in conformation were observed by circular dichroic and infrared spectroscopy. Heparin chromatography also showed the presence of denatured form for these mutants. However, in the presence of multivalent anions such as citrate, sucrose octasulfate, and heparin, the conformation of the mutants resembled that of the wild-type protein, as revealed by X-ray crystallography and circular dichroism spectra of the mutant with a Arg40-->Asp substitution.

Infrared spectroscopic studies of lyophilization- and temperature-induced protein aggregation.

Recent studies have clearly demonstrated that Fourier transform IR spectroscopy can be a powerful tool for the study of protein stabilization during freeze-drying and for optimizing approaches to prevent lyophilization-induced protein aggregation. The purpose of the current review is to provide an overview of these topics, as well as an introduction to the study of protein secondary structure with IR spectroscopy. We will start with a general summary of the theories and practices for processing and interpreting protein IR spectra. We will then review the current literature on the use of IR spectroscopy to study protein structure and the effects of stabilizers during lyophilization. Next we will concentrate specifically on protein aggregation. The bulk of the research and the key assignments of spectral features in protein aggregates come from studies of the effects of high and low temperature on proteins. Therefore, we will first consider this topic. Finally, we will summarize the recent theoretical and applied work on lyophilization-induced aggregation.

Structure and activity of granulocyte colony-stimulating factor derived from CHO cells containing cDNA coding for alternatively spliced sequences.

Two different cDNAs have been isolated, coding for two forms of granulocyte colony-stimulating factor (G-CSF): one for a polypeptide of 174 amino acids and the other for a polypeptide of 177 amino acids. In this paper, we have expressed these two forms in Chinese hamster ovary cells and characterized the purified proteins for activity and conformation. In vitro mitogenic assay showed a 50-fold lower activity for the 177 form than for the 174 form. In vitro receptor binding assay showed that binding of the 177 form to the purified extracellular domain of G-CSF receptor was also diminished, while the 174 form complexed with the receptor. Circular dichroic spectra showed that both forms are similar in the secondary structure, but are slightly different in the tertiary structure. Infrared spectra also showed a slight difference between the two forms. Both techniques also demonstrated differences in stability; i.e., the 174 form is more stable than the 177 form during storage or against heat denaturation.

Some factors associated with the ultrasonic nebulization of proteins.

Ultrasonic nebulization of lactate dehydrogenase (LDH) was investigated using a DeVilbiss "Aerosonic" nebulizer. The enzyme (8ml, 0.025 mg/ml Na2HPO4, pH 7.0) was completely inactivated after 20 minutes of operation. However, the inactivation profile observed during ultrasonic nebulization was different from that previously observed using air-jet nebulization. At least two mechanisms are involved, one associated with heating and the other with aerosol production. By preventing heating of the nebulizer fluid during operation, the denaturation profile was dramatically altered. By additionally including 0.01% w/v Tween 80 or 1% w/v PEG 8000, almost all activity was retained. Similar results were obtained by preventing aerosol production and heating. However, 100% of activity was lost when heating was allowed to occur without aerosol formation. The results demonstrate that cooling in conjunction with a surfactant is one approach that could be used to stabilize proteins to ultrasonic nebulization. However, cooling also significantly reduced solute output from the nebulizer. When operated at 10 degrees C output was negligible. At 50 degrees C the output was 5x greater than that found at room temperature. The median droplet size (micron(s)) was not significantly influenced by the operating temperature of the nebulizer fluid (3.6 +/- 0.4, 21 degrees C; 3.9 +/- 0.2, 50 degrees C, p = NS (n = 6)) although the size distribution was noted to increase at the higher temperature.

High level expression of human leukemia inhibitory factor (LIF) from a synthetic gene in Escherichia coli and the physical and biological characterization of the protein.

LIF is a multi-functional cytokine that elicits effects on a broad range of cell types. In this report, we present the high level expression of human LIF (hLIF) from a chemically synthesized gene template in Escherichia coli where it comprises up to 25% of the cellular protein. The recombinant hLIF, after purification and folding, was examined using CD, FTIR spectroscopy and light scattering. CD and FTIR spectra showed that the hLIF is an alpha-helical protein and has a distinct tertiary structure. The IFTR spectrum resembles that of other four helical bundle proteins including G-CSF and IL-6. Light scattering analysis indicated that it is a monomeric protein, distinguishing it from M-CSF and interferon gamma, which also belong to the class of four helical bundle proteins but are dimeric. Recombinant hLIF was assayed for its activity on the murine leukemic cell line, M-1 as well as on human leukemic cell line, ML-1. It inhibited the growth of M-1 cells and differentiated them towards macrophages. However, it did not have any differentiation inducing effect on human leukemic cell lines alone or in combination with other cytokines.

The conformational stability of a non-covalent dimer of a platelet-derived growth factor-B mutant lacking the two cysteines involved in interchain disulfide bonds.

Native platelet-derived growth factor-B (PDGF-B) forms a covalent dimer through interchain disulfide bonds. In a previous study, an analog of PDGF-B was produced by replacing cysteine 43 and 52, which are involved in the interchain disulfide bonds, with serine. It was revealed that this analog protein has the dimeric molecule weight at pH 4 to 7, forming a non-covalent dimer in solution, and its mitogenic activity is similar to the native covalent dimer. However, the analog protein was more labile to pepsin digestion and low pH treatment, indicating that the interchain disulfides contribute to the stability of the protein. It is interesting to see if the conformation of the protein is affected by elimination of the interchain disulfide bonds, and if the interchain disulfides play any role in the stability of the protein. Circular dichroism and Fourier transform infrared spectroscopic analyses of the analog showed that it has a conformation similar to the wild type at pH 7.5, but is unfolded at pH 2.5, while the native PDGF-B disulfide-linked dimer shows an apparently unaltered conformation at pH 2.5. The analog is also less stable to sodium dodecylsulfate and guanidine HCl-induced denaturation at neutral pH. These results indicate that the non-covalent interactions are sufficient for proper folding and dimer formation at neutral pH, but that the interchain disulfide bonds greatly stabilize the native conformation of PDGF-B.

Infrared spectroscopic studies of calcium binding to inhibited beta-trypsins.

Fourier transform infrared spectroscopy was used to examine the effect of calcium binding on the secondary structure of two inhibited bovine beta-trypsins. Neither the diisopropyl fluorophosphate- nor benzamidine-inhibited forms showed detectable secondary structure perturbation upon calcium binding at pD 6.9 and 5.0, respectively. Considered in light of the recent assignment of an amide I' band to the autolysis loop of bovine beta-trypsin, these results contradict the generally held hypothesis that calcium slows trypsin autolysis by induction of a conformational change at this site and support the recent contention that the mechanism of action has a specific electrostatic origin. In addition, the appearance of a band at 1699 cm-1 in the benzamidine-inhibited form can be interpreted as resulting from the NC-N stretching vibrations of the amidinium moiety, which the observed crystal structure indicates is hydrogen-bonded to the carboxyl group of active-site Asp-189.

Acid-induced unfolding of brain-derived neurotrophic factor results in the formation of a monomeric "a state".

Recombinant human brain-derived neurotrophic factor in acid undergoes a slow loss of tertiary structure as monitored by both near-UV circular dichroism and fluorescence, and appears to retain some secondary structure, as monitored by far-UV circular dichroism and Fourier transform infrared spectroscopy. This loss of tertiary structure parallels a decrease in the weight average molecular weight, from dimer to monomer, when examined using light scattering. Increasing the temperature accelerates this slow reaction. This process may be described most simply as N2 in equilibrium with 2D where N and D are the native and denatured forms of the protein, respectively. However, the acid denaturation strongly depends on the protein concentration, with higher concentration resulting in a lower rate and extent of denaturation. This suggests that the more complicated mechanism N2 in equilibrium with 2N in equilibrium with 2D more accurately describes the denaturation, where the dissociation into a native monomer is the rate-limiting step, and the conversion of N to D occurs relatively rapidly. Size-exclusion chromatography (at neutral pH) at several points during denaturation further demonstrated that the amount of tertiary structure remaining paralleled the dimer concentration and also that the monomer form was long-lived, remaining as monomer during the course of the chromatography. Size-exclusion chromatography and sedimentation velocity determination indicated that the acid-denatured form is a compact molecule. On the basis of the above data, the acid-denatured form may be considered to be a monomeric compact intermediate A state with no tertiary structure but considerable secondary structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Cysteine 17 of recombinant human granulocyte-colony stimulating factor is partially solvent-exposed.

Oh-eda et al. have shown instability of granulocyte-colony stimulating factor (G-CSF) upon storage above pH 7.0 [J. Biol. Chem. (1990) 265, 11,432-11,435]. To clarify the mechanism of this instability, the accessibility of a free cysteinyl residue at position 17 for disulfide exchange reaction was examined using a sulfhydryl reagent. The results show that the cysteine is partially solvent-exposed in both glycosylated and nonglycosylated forms, suggesting that the exposure of the cysteine plays a critical role in the instability of the protein. This is supported by the facts that at low pH where the cysteine is protonated, both proteins have much greater stability and that a Cys17-->Ser analog is extremely stable at neutral pH and 37 degrees C. It was observed that the rate of sulfhydryl titration is slower for the glycosylated form than for the nonglycosylated form, suggesting that the cysteine residue is less solvent-exposed for the former protein or that the pKa is somewhat more basic. In either case, the carbohydrate appears to affect the reactivity of the sulfhydryl group through steric hindrance or alteration in local conformation. Both the glycosylated and nonglycosylated proteins showed essentially identical conformation as determined by circular dichroism, fluorescence, and infrared spectroscopy. Unfolding of these two proteins, induced either by guanidine hydrochloride or by pH, showed an identical course, indicating comparable conformational stability. Contribution of conformational changes to the observed instability at higher pH is unlikely, since little difference in fluorescence spectrum occurs between pH 6.0 and 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)

Dehydration-induced conformational transitions in proteins and their inhibition by stabilizers.

Dehydration of proteins results in significant, measurable conformational changes as observed using Fourier-transform infrared spectroscopy and resolution-enhancement techniques. For several proteins these conformational changes are at least partially irreversible, since, upon rehydration, denaturation and aggregation are observed. The presence of certain stabilizers inhibited these dehydration-induced transitions; the native structure was preserved in the dried state and upon reconstitution. Conformational transitions were also observed in a model polypeptide, poly-L-lysine, after lyophilization and were inhibited with the addition of stabilizing cosolutes. The ability of a particular additive to preserve the aqueous structure of dehydrated proteins and poly-L-lysine upon dehydration correlates directly with its ability to preserve the activity of lactate dehydrogenase, a labile enzyme, during drying.

Production and characterization of an analog of acidic fibroblast growth factor with enhanced stability and biological activity.

We have used recombinant DNA methods to produce two forms of bovine acidic fibroblast growth factor (aFGF), one with alanine substituted for the cysteine at position 47 and the other with the Ala47 change plus the substitution of glycine for the naturally occurring histidine at position 93. Both forms were expressed at high levels in Escherichia coli and purified to near homogeneity by solubilization of the inclusion bodies containing the aFGF, ion exchange chromatography, refolding of the protein and hydrophobic interaction chromatography. Circular dichroic and infrared spectra suggested that the proteins are similar in secondary and tertiary structures and contain little or no alpha-helical conformations. Hydrophobic interaction chromatography showed that aFGF C47A/H93G is slightly more hydrophobic than the aFGF C47A form, suggesting that residue 93 is exposed to the solvent. Half-maximal activity in an in vitro bioassay system was reached at a 10- to 20-fold lower dose for the aFGF C47A/H93G form than for the aFGF C47A form, suggesting that alteration of this residue has an effect on the region responsible for receptor binding. Addition of 50 micrograms/ml heparin enhanced the in vitro activity of the aFGFs, reducing the half-maximal dose to approximately 100 pg/ml for both forms, comparable to that observed previously for basic FGF with or without heparin in this assay system.