Steric Repulsion Forces Contributed by PEGylation of Interleukin-1 Receptor Antagonist Reduce Gelation and Aggregation at the Silicone Oil-Water Interface

Silicone oil, used as a lubricating coating in pharmaceutical containers, has been implicated as a cause of therapeutic protein aggregation. After adsorbing to silicone oil-water interfaces, proteins may form interfacial gels, which can be transported into solution as insoluble aggregates if the interfaces are perturbed. Mechanical interfacial perturbation of both monomeric recombinant human interleukin-1 receptor antagonist (rhIL-1ra) and PEGylated rhIL-1ra (PEG rhIL-1ra) in siliconized syringes resulted in losses of soluble monomeric protein. However, the loss of rhIL-1ra was twice that for PEG rhIL-1ra; even though in solution, PEG rhIL-1ra had a lower ΔG_unf and exhibited a more perturbed tertiary structure at the interface. Net protein-protein interactions in solution for rhIL-1ra were attractive but increased steric repulsion because of PEGylation led to net repulsive interactions for PEG rhIL-1ra. Attractive interactions for rhIL-1ra were associated with increases in intermolecular β-sheet content at the interface, whereas no intermolecular β-sheet structures were observed for adsorbed PEG rhIL-1ra. rhIL-1ra formed interfacial gels that were 5 times stronger than those formed by PEG rhIL-1ra. Thus, the steric repulsion contributed by the PEGylation resulted in decreased interfacial gelation and in the reduction of aggregation, in spite of the destabilizing effects of PEGylation on the protein’s conformational stability.     

Synthesis, characterization and in situ intestinal absorption of different molecular weight scutellarin-PEG conjugates

Highly water soluble esters of scutellarin with different molecular weight polyethylene glycol (PEG) were synthesized. The physicochemical properties, the stabilities under different conditions and the in situ intestinal absorption of the conjugates in rats were investigated. By PEG modification, greatly increased water solubility and a desirable partition coefficient were obtained. These compounds act as prodrugs i.e. breakdown occurrs in a predictable fashion: in vitro, the t1/2 of them in PBS buffer at pH 7.4 was above 12 h (37 degrees C), while in plasma a more rapid breakdown was observed (t1/2 1.5-3 h). PEGylation could enhance the absorption of scutellarin in rat intestine, and scutellarin, its PEG conjugates are absorbed through intestine mainly via passive transport. When the molecular weight of PEG increased from 200 to 1000 Da, the absorption of the conjugates decreased accordingly. The range of PEG molecular weight used for the PEGylation of scutellarin was about 400-1000 Da based on considerations of the yield, the stability and the absorption.     

Effect of the covalent modification with poly(ethylene glycol) on alpha-chymotrypsin stability upon encapsulation in poly(lactic-co-glycolic) microspheres

The effectiveness of the covalent modification of alpha-chymotrypsin with methoxy poly(ethylene glycol) (PEG) to afford its stabilization during encapsulation in poly(lactic-co-glycolic) acid (PLGA) microspheres by a solid-in-oil-in-water method was investigated. alpha-Chymotrypsin was chemically modified with PEG (M(w) = 5000) using molar ratios of PEG-to-chymotrypsin ranging from 0.4 to 96. Various conjugates were obtained and the amount of PEG modification was determined by capillary electrophoresis. In this investigation, only those conjugates with PEG/chymotrypsin molar ratios between approximately 1 and 8 were considered because higher levels of modification caused protein instability even before encapsulation. The stability and functionality of the chymotrypsin formulations were investigated before encapsulation by measuring enzyme kinetics, thermal stability, and tertiary structure intactness, and after the initial lyophilization process by determining the secondary structure content. These stability parameters were related to select ones after encapsulation in PLGA microspheres (specifically, the amount of insoluble aggregates, residual enzyme activity, and magnitude of protein structural perturbations). The results show that the more stable the protein conformation before encapsulation was, the higher was the retention of the specific activity after encapsulation. In contrast, no relationship was found between the protein stability before encapsulation and the magnitude of encapsulation-induced protein aggregation. Even the lowest level of modification (PEG-to-chymotrypsin molar ratio of 0.7) drastically reduced the amount of insoluble aggregates from 18% for the nonmodified protein to 4%. The results demonstrate that PEG modification was able to largely prevent chymotrypsin aggregation and activity loss upon solid-in-oil-in-water encapsulation in PLGA microspheres. It is demonstrated that it is essential to optimize the degree of protein modification to ascertain protein stability upon encapsulation.     

Relating particle formation to salt- and pH-dependent phase separation of non-native aggregates of alpha-chymotrypsinogen a

Visible and subvisible particle formation during the storage of protein solutions is of increasing concern for pharmaceutical products. Previous work (Li Y, Ogunnaike BA, Roberts CJ. 2010. J Pharm Sci 99:645–662) showed that the model protein, alpha‐chymotrypsinogen A (aCgn), forms non‐native aggregates under accelerated (heated) conditions, but the size and morphology of the resulting aggregates depended sensitively on pH and NaCl. Here, it is shown that aggregates created as high‐molecular‐weight soluble aggregates undergo a pH‐ and salt‐dependent reversible phase transition to a condensed or insoluble phase of suspended microparticles, whereas monomers remain completely soluble in the same regime. The location of the phase boundary is quantitatively consistent with the different regimes of kinetic behavior observed previously for aCgn. This suggests that the while kinetics is important for controlling the rates of monomer loss during non‐native aggregation, it may be possible to tune solution thermodynamics and phase behavior to suppress otherwise soluble aggregates from propagating to form visible or large subvisible particles. Interestingly, the aggregate phase boundary is sensitive to the identity of salt anions in solution, highlighting the importance of electrostatics and preferential salt interactions in mediating aggregate condensation and particle formation.     

Population balance modeling of aggregation kinetics of recombinant human interleukin-1 receptor antagonist

The kinetics of benzyl alcohol-induced nonnative aggregation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) were investigated using a population balance model. Steady-state size distributions of rhIL-1ra aggregates formed in a continuous mixed suspension, mixed product removal (MSMPR) reactor were measured and used to extrapolate aggregate nucleation and growth rates parameters. Aggregate growth rate was size-dependent and a linear growth rate model was used to derive a population density function. Addition of 0.9 wt/v% benzyl alcohol increased the nucleation rate by approximately four orders of magnitude. The growth rate for aggregates, however, changed little as a function of benzyl alcohol concentration in the range of 0-0.9%. The addition of sucrose to buffer containing 0.9% benzyl alcohol decreased rhIL1-ra nucleation rate by orders of magnitude and had little impact on growth rate kinetics. The simplicity of the population balance model and the physical relevance of the information obtained from this model render it a useful tool to study protein aggregation kinetics and the effects of excipients on this process.     

Noncovalent PEGylation: different effects of dansyl-, L-tryptophan-, phenylbutylamino-, benzyl- and cholesteryl-PEGs on the aggregation of salmon calcitonin and lysozyme

Protein aggregation is a major instability that can occur during all stages of protein drug production and development. Protein aggregates may compromise the safety and efficacy of the final protein formulation. In this paper, various new excipients [phenylbutylamino-, benzyl-, and cholesteryl-polyethylene glycols (PEGs)] and their use for the reduction of aggregation of salmon calcitonin (sCT) and hen egg-white lysozyme (HEWL) by noncovalent PEGylation are presented. The ability to suppress aggregation of sCT in various buffer systems at a 1:1 molar ratio was assessed by following changes in protein conformation and aggregation state over time. The results are compared with that of dansyl- and L-tryptophan (Trp)-PEGs described in earlier publications. Furthermore, the influence of the different PEG-based excipients on the aggregation of HEWL was measured. HEWL aggregation was completely suppressed in the presence of cholesteryl-PEGs (2 and 5 kDa), whereas deterioration was observed using benzyl-methoxy polyethylene glycols (mPEGs; 2 and 5 kDa). Phenylbutylamino- and Trp-mPEG (2 kDa), as well as dansyl-PEGs of different molecular weight prolonged the lag phase of aggregation and reduced the aggregation velocity of HEWL.     

The relation between moisture‐induced aggregation and structural changes in lyophilized insulin

Objectives Long‐term stability is a critical factor in the successful development of protein pharmaceuticals. Due to the relative instability of proteins in aqueous solutions, they are formulated frequently and stored as lyophilized powders. Exposure of such powders to moisture constitutes a substantial storage problem leading to aggregation and inactivation. We have investigated the structural consequences of moisture sorption by lyophilized insulin under controlled humidity conditions by employing Fourier transform‐infrared (FT‐IR) microscopy. Methods Lyophilized insulin samples were stored in humidity chambers under controlled conditions at 50°C. Protein aggregation studies were carried out by redissolving the insulin samples and measuring the amount of both soluble protein and insoluble aggregates. Near‐UV circular dichroism spectra were collected to assess the tertiary structure. FT‐IR microscopy studies were carried out to investigate secondary structural changes in solid‐state insulin after incubation at different relative humidities. Key findings It was found that sorption of moisture was accompanied by small structural changes in lyophilized insulin at low levels of relative humidity (i.e. 11%). At higher relative humidity levels, structural changes were becoming more pronounced and were characterized by a loss in the α‐helix and increase in β‐sheet content. The magnitude of the structural changes in tendency paralleled the solid‐state instability data (i.e. formation of buffer‐insoluble aggregates and loss in tertiary structure upon reconstitution). Conclusions The results support the hypothesis that water sorption by lyophilized proteins enables structural transitions which can lead to protein aggregation and other deleterious phenomena.     

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.     

Noncovalent pegylation by dansyl-poly(ethylene glycol)s as a new means against aggregation of salmon calcitonin

During all stages of protein drug development, aggregation is one of the most often encountered problems. Covalent conjugation of poly(ethylene glycol) (PEG), also called PEGylation, to proteins has been shown to reduce aggregation of proteins. In this paper, new excipients based on PEG are presented that are able to reduce aggregation of salmon calcitonin (sCT). Several PEG polymers consisting of a hydrophobic dansyl-headgroup attached to PEGs of different molecular weights have been synthesized and characterized physicochemically. After addition of dansyl-methoxypoly(ethylene glycol) (mPEG) 2 kDa to a 40 times molar excess of sCT resulted in an increase in dansyl-fluorescence and a decrease in 90° light scatter suggesting possible interactions. The aggregation of sCT in different buffer systems in presence or absence of the different dansyl-PEGs was measured by changes in Nile red fluorescence and turbidity. Dansyl-mPEG 2 kDa in a 1:1 molar ratio to sCT strongly reduced aggregation. Reduction of sCT aggregation was also measured for the bivalent dansyl-PEG 3 kDa in a 1:1 molar ratio. Dansyl-mPEG 5 kDa deteriorated sCT aggregation. Potential cytotoxicity and hemolysis were investigated. This paper shows that dansyl-PEGs are efficacious in reducing aggregation of sCT.     

Influence of PEGylation with linear and branched PEG chains on the adsorption of glucagon to hydrophobic surfaces

PEGylation has proven useful for prolonging the plasma half lives of proteins, and since approval of the first PEGylated protein drug product by the FDA in 1990, several PEGylated protein drug products have been marketed. However, the influence of PEGylation on the behavior of proteins at interfaces is only poorly understood. The aim of this work was to study the effect of PEGylation on the adsorption of glucagon from aqueous solution to a hydrophobic surface and to compare the effects of PEGylation with a linear and a branched PEG chain, respectively. The 3483 Da peptide glucagon was PEGylated with a 2.2 kDa linear and a branched PEG chain, respectively, and the adsorption behaviors of the three proteins were compared using isothermal titration calorimetry, fixed-angle optical reflectometry and total internal reflection fluorescence. PEGylation decreased the number of glucagon molecules adsorbing per unit surface area and increased the initial adsorption rate of glucagon. Furthermore, the results indicated that the orientation and/or structural changes of glucagon upon adsorption were affected by the PEGylation. Finally, from the isothermal titration calorimetry and the reflectometry data, it was observed that the architecture of the PEG chains had an influence on the observed heat flow upon adsorption as well as on the initial rate of adsorption, respectively.     

Effects of pH, temperature, and sucrose on benzyl alcohol-induced aggregation of recombinant human granulocyte colony stimulating factor

Antimicrobial preservatives (e.g., benzyl alcohol), which are required in multidose formulations, can induce protein aggregation. In this study, the mechanism of benzyl alcohol-induced aggregation of recombinant human granulocyte colony-stimulating factor (rhGCSF) was investigated by determining the effects of temperature, pH, and sucrose on this process. rhGCSF was incubated at 25 and 37 degrees C and at pH 7.0 (phosphate-buffered saline, PBS) and pH 3.5 (HCl). Benzyl alcohol (0.9% w/v) accelerated aggregation of rhGCSF at pH 7.0, an effect that was much greater at 37 degrees C than at 25 degrees C and partially counteracted by 1.0 M sucrose. At pH 3.5, benzyl alcohol did not induce aggregation of rhGCSF. Spectroscopic studies showed that 0.9% benzyl alcohol altered the tertiary structure of rhGCSF at both pH, without detectably altering secondary structure. Structural perturbation was greater at 37 degrees C than at 25 degrees C. At both pH 7.0 and 3.5, the hydrogen-deuterium (H-D) exchange rate for rhGCSF was increased by 0.9% benzyl alcohol. Sucrose (1.0 M) partially counteracted the benzyl alcohol-induced perturbation of tertiary structure and the increase in H-D exchange rate. Thus, benzyl alcohol accelerates aggregation of rhGCSF at pH 7.0, because it favors partially unfolded aggregation-prone conformations of the protein. Sucrose partially counteracts benzyl alcohol-induced rhGCSF aggregation by shifting the molecular population away from these species and towards more compact conformations. We postulate that the absence of aggregation at pH 3.5, even with benzyl alcohol-induced structural perturbation, is due to the unfavorable energetics of intermolecular interactions (i.e., colloidal stability) between rhGCSF molecules at this pH.     

Effects of PEG size on structure, function and stability of PEGylated BSA

The effects of PEGylation on the structural, thermal and functional stability of bovine serum albumin (BSA) were investigated using BSA and 6 linear mono-PEGylated BSA compounds. The secondary and tertiary structure of BSA measured by circular dichroism (CD) was independent of PEGylation. In contrast, the thermal stability of BSA was affected by PEGylation. The apparent unfolding temperature T_max measured by differential scanning calorimetry (DSC) decreased with PEGylation, whereas the temperature of aggregation, T_agg, measured by dynamic light scattering (DLS) increased with PEGylation. The unfolding temperature and the temperature of aggregation were both independent of the molecular weight of the PEG chain. Possible functional changes of BSA after PEGylation were measured by Isothermal Titration Calorimetry (ITC), where the binding of sodium dodecyl sulphate (SDS) to BSA and PEGylated BSA was analysed. At 25 °C, two distinct classes of binding sites (high affinity and low affinity) for BSA and one class of binding site (low affinity) for PEGylated BSA were identified. The binding isotherm was modelled assuming independence and thermodynamic equivalence of the sites within each class. From the present biophysical characterisation, it is concluded that after PEGylation BSA appears to be unaffected structurally (secondary and tertiary structure), slightly destabilised thermally (unfolding temperature), stabilised kinetically (temperature of aggregation) and has an altered functionality (binding profile). These biophysical characteristics are all independent of the molecular weight of the attached polymer chain.     

Role of benzyl alcohol in the prevention of heat-induced aggregation and inactivation of hen egg white lysozyme

The aim of the study was to investigate the stability of a model protein, lysozyme, in the presence of the commonly used preservative benzyl alcohol. Techniques including lytic assay, size exclusion chromatography, circular dichroism, differential scanning calorimetry, native polyacrylamide gel electrophoresis and dynamic light scattering were used to study the overall stability of lysozyme in the presence of benzyl alcohol. The stability of lysozyme against thermal stress was higher in the presence of benzyl alcohol. In the presence of 0.5%, 0.9% and 2% v/v benzyl alcohol, the enzyme showed 33%, 42% and 75% residual activity, respectively, when exposed to 75 °C for 2 h, as compared to the 22% activity of control sample. A gradual increase in the size of aggregates was observed for the control sample relative to the samples containing benzyl alcohol, as a result of loss of monomer concentration. The effect was found to be concentration-dependent with 2% benzyl alcohol showing maximum prevention of heat-induced unfolding and aggregation. This effect is remarkable since the thermal transition temperature of the enzyme decreases in the presence of benzyl alcohol. Benzyl alcohol favours the thermal denaturation of lysozyme but stabilizes the lysozyme against the heat-induced aggregation.     

The Effect of Benzyl Alcohol on Recombinant Human Interferon-γ

Purpose. The goal of this study was to investigate the conformational change and aggregation of recombinant human interferon-gamma (rhIFN-) as a result of interaction between benzyl alcohol and the protein. The effects of buffer concentration, buffer species, ionic strength, rhIFN- and benzyl alcohol concentrations on the dynamics of the interaction in liquid formulations were also examined. Methods. The effect of benzyl alcohol on the secondary and tertiary structure of rhIFN- in succinate and acetate buffers was studied using far-UV and near-UV circular dichroism spectrophotometry, respectively. Dynamic light scattering was employed to detect aggregate formation due to the interaction of benzyl alcohol with rhIFN-. Results. The addition of benzyl alcohol at 0.9% (w/v) in various liquid rhIFN- formulations induced changes in circular dichroism (CD) spectra of the protein in the near-UV region, while the CD spectra in the far-UV region remained unaltered. There were gradual decreases in ellipticity with time throughout the near-UV CD spectra. The decreases in near-UV ellipticity induced by benzyl alcohol were accompanied by the formation of high molecular weight aggregates as measured by dynamic light scattering. Loss in near-UV ellipticity was accelerated at lower protein concentration and by increasing buffer or benzyl alcohol concentration. It was also faster in succinate than in acetate buffer. Formulation ionic strength did not affect the CD spectral changes in both the near- and far-UV regions. Conclusions. Interaction between benzyl alcohol and rhIFN- is formulation dependent. Protein concentration, buffer species, buffer concentration, and preservative concentration play a significant role in determining the extent of the interaction and consequently the stability of the product.     

Effect of PEGylation on stability of peptide in poly(lactide-co-glycolide) microspheres

The purpose of this study was to evaluate the effect of PEGylation on the stabilization of peptide in poly(D,L-lactide-co-glycolide) (PLGA) microspheres for sustained release delivery. As model peptide, growth hormone-releasing peptide-6 (GHRP-6) was conjugated with succinimidyl propionate monomethoxy poly(ethylene glycol) (PEG) with an average molecular weight of 2000 Da. The mono-PEG-GHRP-6 was separated by ion-exchange chromatography, and its molecular mass was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The microspheres encapsulating native GHRP-6 or mono-PEG-GHRP-6 were prepared using the single oil-in-water emulsion solvent evaporation method. During incubation in a 0.1 M phosphate buffer (pH 7.4) for one month at 37°C, native GHRP-6 microspheres were identified to form several acylated peptides by reversed-phase HPLC and MALDI-TOF MS, whereas the mono-PEG-GHRP-6 microspheres was not affected from peptide acylation by PLGA. This study demonstrates that PEGylation can stabilize peptide against the acylation reaction occurred in PLGA microspheres.     

Stabilization of Lysozyme by Benzyl Alcohol: Surface Tension and Thermodynamic Parameters

The aim of the study was to understand the effect of benzyl alcohol on biological activity, aggregation behavior, denaturant and heat-induced unfolding of lysozyme. Compatibility studies of lysozyme carried out with a number of anti-microbial preservatives, indicated benzyl alcohol to be the best suppressor of protein aggregation against heat stress. The effect of this preservative was checked at various pH values ranging from 4.0 to 9.0. In spite of reducing the thermal denaturation temperature (T_m) at all pH values, benzyl alcohol had a stabilizing effect on lysozyme in terms of retaining the biological activity when the enzyme was incubated at 75°C. The reduction in T_m with increasing benzyl alcohol concentration was correlated with decreasing surface tension of surrounding medium. A detailed thermodynamic study of lysozyme in the presence of benzyl alcohol was carried out at pH 6.2. Change in Gibb’s free energy of thermal unfolding at 25°C was found to remain constant in the presence of benzyl alcohol, indicating no interaction of benzyl alcohol with the native protein at room temperature. Both the enthalpy and entropy change at mid point of thermal unfolding were found to increase in the presence of benzyl alcohol indicating the stabilization of partially unfolded state.     

Complement activation by PEG-functionalized multi-walled carbon nanotubes is independent of PEG molecular mass and surface density

Carboxylated (4%) multi-walled carbon nanotubes were covalently functionalized with poly(ethylene glycol)₁₀₀₀ (PEG₁₀₀₀), PEG₁₅₀₀ and PEG₄₀₀₀ with a PEG loading of approximately 11% in all cases. PEG loading generated non-uniform and heterogeneous higher surface structures and increased nanotube width considerably, but all PEGylated nanotube species activated the complement system in human serum equally. Increased PEG loading, through adsorption of methoxyPEG_{2000(or 5000)}-phospholipid conjugates, generated fewer complement activation products; however, complement activation was never completely eliminated. Our observations address the difficulty in making carbon nanotubes more compatible with innate immunity through covalent PEG functionalization as well as double PEGylation strategies.From the Clinical EditorComplement-mediated toxicity is a major limiting factor in certain nanomedicine applications. This study clarifies that PEGylation of carbon nanotubes is unlikely to address this complication.     

Mechanism for benzyl alcohol-induced aggregation of recombinant human interleukin-1 receptor antagonist in aqueous solution

Benzyl alcohol, an antimicrobial preservative, accelerates aggregation and precipitation of recombinant human interleukin-1 receptor antagonist (rhIL-1ra) in aqueous solution. The loss of native monomer during incubation at 37 degrees C was determined by analysis of sample aliquots with size exclusion high performance liquid chromatography (SE-HPLC). Benzyl alcohol caused minor perturbation of the tertiary structure of the protein without changing its secondary structure, documenting that the preservative caused a minor shift in the protein molecular population toward partially unfolded species. Consistent with this conclusion, in the presence of benzyl alcohol the rate of H-D exchange was accelerated and the fluorescence of 1-anilinonaphthalene-8-sulfonic acid in the presence of rhIL1ra was increased. Benzyl alcohol did not alter the free energy of unfolding based on unfolding experiments in urea or guanidine HCl. With differential scanning calorimetry it was determined that benzyl alcohol reduced the apparent Tm of rhIL-1ra, but this effect occurred because the preservative lowered the temperature at which the protein aggregated during heating. Isothermal calorimetry documented that the interaction of benzyl alcohol with rhIL-1ra is relatively weak and hydrophobically driven. Thus, benzyl alcohol accelerates protein aggregation by binding to the protein and favoring an increase in the level of partially unfolded, aggregation-competent species. Sucrose partially inhibited benzyl alcohol-induced aggregation and tertiary structural change. Sucrose is preferentially excluded from the surface of the protein, favoring most compact native state species over expanded aggregation-prone forms.     

Effects of benzyl alcohol on aggregation of recombinant human interleukin-1-receptor antagonist in reconstituted lyophilized formulations

A major limitation in the successful development of multidose protein formulations is protein aggregation induced by antimicrobial preservatives such as benzyl alcohol, which are included to maintain product sterility. Studies were conducted to evaluate the strategy of developing lyophilized formulations of a therapeutic protein, recombinant human interlukin-1 receptor antagonist (rhIL-1ra), to be reconstituted with a bacteriostatic amount (0.9% w/v) of benzyl alcohol in water. The strategy was based on the following hypotheses. The first was that benzyl alcohol would foster aggregation during reconstitution of the lyophilized sample. The second hypothesis was that the extent of benzyl alcohol-induced protein aggregation would correlate directly with the degree of structural perturbation of rhIL-1ra in the dried solid after lyophilization. Differential structural retention of rhIL-1ra in the dried solid was obtained by using a combination of formulation variables important for lyophilization and included: protein concentration, type of stabilizer, and presence or absence of NaCl. Infrared spectroscopic analysis of the lyophilized samples indicated that high initial solution protein concentration and the stabilizer sucrose minimized structural perturbation of rhIL-1ra during lyophilization. In contrast, NaCl was destabilizing. Reconstitution of the dried solid with 0.9% (w/v) benzyl alcohol caused a greater degree of protein aggregation than reconstitution with water, confirming our first hypothesis. In support of our second hypothesis, the extent of aggregation induced by benzyl alcohol during reconstitution was strongly modulated by the degree of retention of native rhIL-1ra secondary structure during lyophilization. During storage of the reconstituted lyophilized samples at room temperature, benzyl alcohol did not accelerate aggregation of rhIL-1ra. This study demonstrated that for development a multidose lyophilized protein formulation involving reconstitution with a solution of benzyl alcohol, protein structural perturbations during freeze-drying should be minimized with a stabilizing excipient and appropriate choice of protein concentration and tonicity modifier. Furthermore, postreconstitution storage at reduced temperature (e.g., room temperature or 4 degrees C) could minimize the risk of preservative-induced protein aggregation.     

Techniques for Assessing the Effects of Pharmaceutical Excipients on the Aggregation of Porcine Growth Hormone

Three denaturing techniques have been evaluated for their ability to induce irreversible aggregation and precipitation of recombinant porcine growth hormone (pGH). The denaturing stimuli included thermal denaturation, interfacial denaturation through the introduction of a high air/water interface by vortex agitation, and a guanidine (Gdn) HC1 technique which involved rapid dilution of a partially unfolded state of pGH to nondenaturing conditions. Soluble and insoluble pGH fractions were evaluated for the presence of covalently modified species and soluble aggregates by size exclusion chromatography (SEC), sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE), and isoelectric focusing (IEF). In each of the three denaturation methods, precipitation was found to be irreversible, as the precipitated pellet could not be solubilized upon resuspending in buffer. The soluble pGH fractions consisted of only monomeric material and the insoluble protein pellet could be completely solubilized with Gdn HC1 or SDS. There was no evidence of detectable covalent modifications in the precipitated protein pellet following any of the three denaturation techniques. Three excipients, Tween 20, hydroxypropyl--cyclodextrin (HPCD), and sorbitol were evaluated for their stabilizing ability using each of the three denaturation methods and the degree of stabilization was found to be dependent upon the denaturing stimulus incorporated. Tween 20 was found to be highly effective in preventing pGH precipitation using the interfacial and Gdn techniques and was moderately effective using the thermal denaturation method. Inclusion of HPCD in the sample buffer significantly reduced precipitation using the thermal and interfacial methods but was ineffective in the Gdn technique. In contrast, sorbitol was ineffective in the interfacial technique and only moderately effective at high concentrations in reducing Gdn- and thermally-induced precipitation. These studies demonstrate the need to consider the nature of the denaturing stimulus when evaluating the potential protein-stabilizing properties of different pharmaceutical excipients.