RESUMO
PURPOSE: To determine the effect of solution conditions, especially low ionic strength, on the dynamics of molecular diffusion and protein-protein interactions in monoclonal antibody solutions. METHODS: The interaction parameter, kD, was calculated from diffusion data obtained from dynamic light scattering (DLS) measurements performed using a Zetasizer. Theoretical considerations were utilized to evaluate the hard sphere and electrostatic contribution to molecular interactions. RESULTS: At low ionic strengths, repulsions were the dominant forces governing the behavior of both mAbs. As ionic strength increased, attractions contributed to the behavior of mAb1, while repulsions remained the dominant factor affecting mAb3 behavior. Repulsions alone were not sufficient to affect mAb3 viscosity in water, while the presence of repulsions as well as specific attractions was suggested to cause an increase in the viscosity of mAb1 in water compared to 15 mM ionic strength. CONCLUSIONS: Solution physical properties varied for the mAbs investigated. Our findings highlighted the importance of developing a fundamental understanding of interplay of forces governing solution properties of each individual mAb under low ionic strength conditions. Such understanding is critical in enabling successful development of self-buffered formulations.
Assuntos
Anticorpos Monoclonais/química , Soluções Tampão , Química Farmacêutica , Difusão , Concentração de Íons de Hidrogênio , Concentração Osmolar , Espalhamento de Radiação , Soluções/química , ViscosidadeRESUMO
PURPOSE: To determine the intrinsic viscosity of several monoclonal antibodies (mAbs) under varying pH and ionic strength solution conditions. METHODS: An online viscosity detector attached to HPLC (Viscotek®) was used to determine the intrinsic viscosity of mAbs. The Ross and Minton equation was used for viscosity prediction at high protein concentrations. Bulk viscosity was determined by a Cambridge viscometer. RESULTS: At 15 mM ionic strength, intrinsic viscosity of the mAbs determined by the single-point approach varied from 5.6 to 6.4 mL/g with changes in pH. High ionic strength did not significantly alter intrinsic viscosity, while a significant increase (up to 24.0 mL/g) was observed near zero mM. No difference in bulk viscosity of mAb3 was observed around pH 6 as a function of ionic strength. Data analysis revealed that near zero mM ionic strength limitations of the single-point technique result in erroneously high intrinsic viscosity. CONCLUSIONS: Intrinsic viscosity is a valuable tool that can be used to model baseline viscosity at higher protein concentrations. However, it is not predictive of solution non-ideality at higher protein concentrations. Furthermore, breakdown of numerous assumptions limits the applicability of experimental techniques near zero mM ionic strength conditions. For molecules and conditions studied, the single-point approach produced reliable intrinsic viscosity results at 15 mM. However, this approach must be used with caution near zero mM ionic strength. Data analysis can be used to reveal whether determined intrinsic viscosity is reliable or erroneously high.
Assuntos
Anticorpos Monoclonais/química , Soluções/química , Química Farmacêutica , Cromatografia Líquida de Alta Pressão , Humanos , Concentração de Íons de Hidrogênio , Concentração Osmolar , Solubilidade , ViscosidadeRESUMO
PURPOSE: To systematically analyze shape and size of soluble irreversible aggregates and the effect of aggregate formation on viscosity. METHODS: Online light scattering, refractive index and viscosity detectors attached to HPLC (Viscotek®) were used to study aggregation, molecular weight and intrinsic viscosity of bovine serum albumin (BSA). Irreversible aggregates were generated by heat stress. Bulk viscosity was measured by an oscillating piston viscometer. RESULTS: As BSA was heated at a higher concentration or for a longer time, the relative contribution, molecular weight and intrinsic viscosity of aggregate species increased. Molecular shape was evaluated from intrinsic viscosity values, and aggregates were estimated to be more asymmetric than monomer species. The presence of aggregates resulted in an increase in bulk viscosity when relative contribution of very high molecular weight species exceeded 10%. CONCLUSIONS: For model system and conditions studied, generation of higher order aggregate species was concluded to be associated with an increase in molecular asymmetry. Elevated viscosity in the presence of aggregated species points to molecular asymmetry being a critical parameter affecting solution viscosity of BSA.
Assuntos
Soroalbumina Bovina/química , Animais , Bovinos , Cromatografia Líquida de Alta Pressão , Difusão Dinâmica da Luz , Temperatura Alta , Hidrodinâmica , Concentração de Íons de Hidrogênio , Estrutura Molecular , Peso Molecular , Agregados Proteicos , Refratometria , ViscosidadeRESUMO
Opalescence in protein solutions reduces aesthetic appeal of a formulation and can be an indicator of the presence of aggregates or precursor to phase separation in solution signifying reduced product stability. Liquid-liquid phase separation of a protein solution into a protein-rich and a protein-poor phase has been well-documented for globular proteins and recently observed for monoclonal antibody solutions, resulting in physical instability of the formulation. The present review discusses opalescence and liquid-liquid phase separation (LLPS) for therapeutic protein formulations. A brief discussion on theoretical concepts based on thermodynamics, kinetics, and light scattering is presented. This review also discusses theoretical concepts behind intense light scattering in the vicinity of the critical point termed as "critical opalescence". Both opalescence and LLPS are affected by the formulation factors including pH, ionic strength, protein concentration, temperature, and excipients. Literature reports for the effect of these formulation factors on attractive protein-protein interactions in solution as assessed by the second virial coefficient (B2) and the cloud-point temperature (Tcloud) measurements are also presented. The review also highlights pharmaceutical implications of LLPS in protein solutions.
Assuntos
Iridescência/efeitos dos fármacos , Soluções Farmacêuticas/química , Soluções Farmacêuticas/farmacologia , Proteínas/química , Soluções/química , Soluções/farmacologia , Anticorpos Monoclonais/química , Química Farmacêutica/métodos , Humanos , Concentração de Íons de Hidrogênio , Concentração Osmolar , Temperatura , TermodinâmicaRESUMO
Liquid-liquid phase separation (LLPS) and aggregation can reduce the physical stability of therapeutic protein formulations. On undergoing LLPS, the protein-rich phase can promote aggregation during storage due to high concentration of the protein. Effect of different excipients on aggregation in protein solution is well documented; however data on the effect of excipients on LLPS is scarce in the literature. In this study, the effect of four excipients (PEG 400, Tween 80, sucrose, and hydroxypropyl beta-cyclodextrin (HPßCD)) on liquid-liquid phase separation and aggregation in a dual variable domain immunoglobulin protein solution was investigated. Sucrose suppressed both LLPS and aggregation, Tween 80 had no effect on either, and PEG 400 increased LLPS and aggregation. Attractive protein-protein interactions and liquid-liquid phase separation decreased with increasing concentration of HPßCD, indicating its specific binding to the protein. However, HPßCD had no effect on the formation of soluble aggregates and fragments in this study. LLPS and aggregation are highly temperature dependent; at low temperature protein exhibits LLPS, at high temperature protein exhibits aggregation, and at an intermediate temperature both phenomena occur simultaneously depending on the solution conditions.
Assuntos
Excipientes/farmacologia , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/metabolismo , Excipientes/química , Humanos , Extração Líquido-Líquido , Soluções Farmacêuticas , Transição de Fase , Domínios e Motivos de Interação entre Proteínas , Multimerização ProteicaRESUMO
PURPOSE: Increased solution viscosity results in difficulties in manufacturing and delivery of therapeutic protein formulations, increasing both the time and production costs, and leading to patient inconvenience. The solution viscosity is affected by the molecular properties of both the solute and the solvent. The purpose of this work was to investigate the effect of size, charge and protein-protein interactions on the viscosity of Dual Variable Domain Immunoglobulin (DVD-Ig(TM)) protein solutions. METHODS: The effect of size of the protein molecule on solution viscosity was investigated by measuring intrinsic viscosity and excluded volume calculations for monoclonal antibody (mAb) and DVD-Ig(TM) protein solutions. The role of the electrostatic charge resulting in electroviscous effects for DVD-Ig(TM) protein was assessed by measuring zeta potential. Light scattering measurements were performed to detect protein-protein interactions affecting solution viscosity. RESULTS: DVD-Ig(TM) protein exhibited significantly higher viscosity compared to mAb. Intrinsic viscosity and excluded volume calculations indicated that the size of the molecule affects viscosity significantly at higher concentrations, while the effect was minimal at intermediate concentrations. Electroviscous contribution to the viscosity of DVD-Ig(TM) protein varied depending on the presence or absence of ions in the solution. In buffered solutions, negative k D and B 2 values indicated the presence of attractive interactions which resulted in high viscosity for DVD-Ig(TM) protein at certain pH and ionic strength conditions. CONCLUSIONS: Results show that more than one factor contributes to the increased viscosity of DVD-Ig(TM) protein and interplay of these factors modulates the overall viscosity behavior of the solution, especially at higher concentrations.
Assuntos
Imunoglobulinas Intravenosas/química , Viscosidade , Algoritmos , Anticorpos Monoclonais/química , Soluções Tampão , Humanos , Concentração de Íons de Hidrogênio , Peso Molecular , Soluções Farmacêuticas , Proteínas/química , Soluções , Eletricidade EstáticaRESUMO
Dual variable domain immunoglobulin proteins (DVD-Ig proteins) are large molecules (MW â¼ 200 kDa) with increased asymmetry because of their extended Y-like shape, which results in increased formulation challenges. Liquid-liquid phase separation (LLPS) of protein solutions into protein-rich and protein-poor phases reduces solution stability at intermediate concentrations and lower temperatures, and is a serious concern in formulation development as therapeutic proteins are generally stored at refrigerated conditions. In the current work, LLPS was studied for a DVD-Ig protein molecule as a function of solution conditions by measuring solution opalescence. LLPS of the protein was confirmed by equilibrium studies and by visually observing under microscope. The protein does not undergo any structural change after phase separation. Protein-protein interactions were measured by light scattering (kD) and Tcloud (temperature that marks the onset of phase separation). There is a good agreement between kD measured in dilute solution with Tcloud measured in the critical concentration range. Results indicate that the increased complexity of the molecule (with respect to size, shape, and charge distribution on the molecule) increases contribution of specific and nonspecific interactions in solution, which are affected by formulation factors, resulting in LLPS for DVD-Ig protein.
Assuntos
Imunoglobulina G/química , Imunoglobulina G/metabolismo , Região Variável de Imunoglobulina/química , Região Variável de Imunoglobulina/metabolismo , Soluções Farmacêuticas/química , Transição de Fase , Domínios e Motivos de Interação entre Proteínas , Química Farmacêutica , Humanos , Concentração de Íons de Hidrogênio , Concentração Osmolar , Soluções , TemperaturaRESUMO
PURPOSE: The purpose of this study was to investigate the contribution of the dipole moment to overall protein-protein interactions and viscosity of a monoclonal antibody MAb1. METHODS: The dipole moment of MAb1 was measured at various solution pH conditions using dielectric relaxation spectroscopy. RESULTS: The dipole moment for MAb1 was highest at pH 6.5, and the pH dependent change in molecular dipole correlated fairly well with previously observed trends of viscosity and storage modulus versus pH. Moreover, the magnitude of the dielectric increment at pH 6.5 and 7.0 showed strong concentration dependence, indicating the presence of relatively strong dipole-dipole interactions at these pHs. To test if the cluster of charged residues present in the Fab contributes to the mean dipole moment observed for MAb1, additional mutants involving charge mutations in the CDR were investigated. In contrast to MAb1, all of the other MAbs showed significantly reduced pH and concentration dependence of the measured dipole moments and dielectric increments, respectively. CONCLUSIONS: The solution pH dependent measured dipole moments of MAb1 appears to be in line with the observed intermolecular interactions and viscosity behavior suggesting that dipole-dipole interaction plays an important role in governing the high concentration solution behavior of this MAb.
Assuntos
Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Espectroscopia Dielétrica , Concentração de Íons de Hidrogênio , Modelos Moleculares , Mapas de Interação de Proteínas , Eletricidade Estática , ViscosidadeRESUMO
PURPOSE: To study the effect of three nonionic surfactants on the protein-silicone oil interactions. METHODS: The adsorption of Tween® 80, Pluronic® F68 and Tween® 20 at the silicone oil/water interface (using shifts in frequency (ΔF) and resistance (ΔR) with quartz crystal microbalance) was compared to the adsorption at air/water interface (using surface tension). Effect of surfactants on protein adsorption to the silicone oil/water interface was studied in sequential- and co-adsorption modes. Protein-surfactant binding in the bulk was measured using dynamic surface tension method. RESULTS: Saturation of air/water and silicone oil/water interfaces by surfactants was observed at similar bulk concentrations. ΔF due to protein adsorption to the interface decreased only when surfactant was present as a pre-adsorbed species. Insignificant differences in the dynamic surface tension values of surfactant solutions were observed in the presence of protein. CONCLUSIONS: Similar hydrophobic forces were responsible for driving the surfactant adsorption at both air/water and silicone oil/water interfaces. Surfactants were effective in reducing the protein adsorption to the silicone oil only when introduced before or along with the protein. No significant binding between the protein and surfactants was observed in the bulk.
Assuntos
Poloxâmero/química , Polissorbatos/química , Proteínas Recombinantes de Fusão/química , Óleos de Silicone/química , Tensoativos/química , Adsorção , Elasticidade , Propriedades de Superfície , Tensão Superficial , ViscosidadeRESUMO
In this study, we report the effects of acidic to basic residue point mutations (5K) on the dipole moment of RNAse SA at different pHs. Dipole moments were determined by measuring solution capacitance of the wild type (WT) and the 5K mutant with an impedance analyzer. The dipole moments were then (1) compared with theoretically calculated dipole moments, (2) analyzed to determine the effect of the point mutations, and (3) analyzed for their contribution to overall protein-protein interactions (PPI) in solution as quantitated by experimentally derived second virial coefficients. We determined that experimental and calculated dipoles were in reasonable agreement. Differences are likely due to local motions of residue side chains, which are not accounted for by the calculated dipole. We observed that the proteins' dipole moments increase as the pH is shifted further from their isoelectric points and that the wild-type dipole moments were greater than those of the 5K. This is likely due to an increase in the proportion of one charge (either negative or positive) relative to the other. A greater charge disparity corresponded to a larger dipole moment. Finally, the larger dipole moments of the WT resulted in greater attractive overall PPI for that protein as compared to the 5K.
Assuntos
Simulação de Dinâmica Molecular , Proteínas Mutantes/química , Ribonucleases/química , Eletricidade Estática , Capacitância Elétrica , Concentração de Íons de Hidrogênio , Lisina/química , Concentração Osmolar , Mutação Puntual , Mapeamento de Interação de Proteínas , Estrutura Terciária de Proteína , Ribonucleases/genética , Solubilidade , Soluções/químicaRESUMO
The present work investigates the influence of electrostatic surface potential distribution of monoclonal antibodies (MAbs) on intermolecular interactions and viscosity. Electrostatic models suggest MAb-1 has a less uniform surface charge distribution than MAb-2. The patches of positive and negative potential on MAb-1 are predicted to favor intermolecular attraction, even in the presence of a small net positive charge. Consistent with this expectation, MAb-1 exhibits a negative second virial coefficient (B22), an increase in static structure factor, S((qâ0)), and a decrease in hydrodynamic interaction parameter, H((qâ0)), with increase in MAb-1 concentration. Conversely, MAb-2 did not show such heterogeneous charge distribution as MAb-1 and hence favors intermolecular repulsion (positive B22), lower static structure factor, S((qâ0)), and repulsion induced increase in momentum transfer, H((qâ0)), to result in lower viscosity of MAb-2. Charge swap mutants of MAb-1, M-5 and M-7, showed a decrease in charge asymmetry and concomitantly a loss in self-associating behavior and lower viscosity than MAb-1. However, replacement of charge residues in the sequence of MAb-2, M-10, did not invoke charge distribution to the same extent as MAb-1 and hence exhibited a similar viscosity and self-association profile as MAb-2.
Assuntos
Anticorpos Monoclonais/química , Soluções/química , Eletricidade Estática , ViscosidadeRESUMO
PURPOSE: To investigate the physical stability of antibody-polyol formulations under thermal and mechanical stresses. METHODS: mAb-U was analyzed in buffer, trehalose, sucrose, glycerol and ethylene glycol solutions at pH 7.0. T(m1) of mAb-U was determined using DSC. Thermal stress studies were performed by incubating mAb-U-polyol solutions at 40°C (2 months), 50°C (3 weeks) and 65°C (5 days). Mechanical stress studies were conducted by shaking mAb-U-polyol solutions at 200 rpm for 5 days at 25°C. RESULTS: Trehalose and glycerol increased the T(m1) of mAb-U, whereas ethylene glycol decreased it. The trend observed in the order of increasing aggregation of mAb-U after thermal stress (40°C and 50°C) was buffer = trehalose = sucroseAssuntos
Anticorpos Monoclonais/química
, Adsorção
, Etilenoglicol/química
, Conformação Proteica
, Estabilidade Proteica
, Desdobramento de Proteína
, Solubilidade
, Estresse Mecânico
, Sacarose/química
, Temperatura
, Trealose/química
RESUMO
Addition of pharmaceutical excipients is a commonly used approach to decrease the viscosity of highly concentrated protein formulations, which otherwise could not be subcutaneously injected or processed. The variety of protein-protein interactions, which are responsible for increased viscosities, makes a portfolio approach necessary. Screening of several excipients to develop such a portfolio is time and money consuming in industrial settings. Responsible protein-protein interactions were investigated using the interaction parameter kD obtained from dynamic light scattering measurements in the studies presented herein. Together with in-silico calculated excipient parameter, kD could be used as a screening tool accelerating screening and formulation development as kD is suitable to high-throughput formats using small quantities of protein and low concentrations. A qualitative correlation between kD and high-concentration viscosity behavior could be shown in our case.
Assuntos
Excipientes , Imunoglobulina G , Anticorpos Monoclonais , Desenvolvimento Industrial , ViscosidadeRESUMO
The present work discusses an alternative procedure to obtain static light scattering (SLS) parameters in a dilute and semidilute concentration regime from a dynamic light scattering (DLS) instrument that uses an avalanche photodiode (APD) for recording the scattered intensity signal. An APD enables one to perform both SLS and DLS measurements by photon counting and photon correlation, respectively. However, due to the associated recovery time, the APDs are susceptible to saturation (above 1000 kcps), which may limit the measurements in systems that scatter too much light. We propose an alternative way of obtaining the SLS parameters with instruments that use APD for recording signal intensities.
Assuntos
Luz , Espalhamento de Radiação , Anticorpos Monoclonais/química , Quimotripsinogênio/metabolismo , Difusão , Concentração de Íons de Hidrogênio , Imunoglobulina G/químicaRESUMO
PURPOSE: To understand the apparent inconsistency between the dilute and high concentration viscosity behavior of bovine serum albumin (BSA). METHOD: Zeta potential and molecular charge on BSA were determined from Electrophoretic mobility measurements. Second virial coefficient (B(22)) and interaction parameter (k(D)) obtained from static and dynamic light scattering, respectively, quantified intermolecular interactions. Rheology studies characterized viscoelasticity at high concentration. The dipole moment was calculated using Takashima's approximation for proton fluctuations over charged residues. RESULTS: The effective isoelectric point of BSA was pH 4.95. In dilute solutions (≤ 40 mg/ml), the viscosity was minimal at the pI; at high concentrations, pH 5.0 solutions were most viscous. B(22) and k(D) showed intermolecular attractions at pH 5.0; repulsions dominated at other pHs. The attractive interactions led to a high storage modulus (G') at pH 5.0. CONCLUSION: In dilute solutions, the electroviscous effect due to net charge governs the viscosity behavior; at high concentrations, the solution viscosity cannot be justified based on a single parameter. The net interplay of all intermolecular forces dictates viscosity behavior, wherein intermolecular attraction leads to a higher solution viscosity.
Assuntos
Soroalbumina Bovina/química , Soluções/química , Animais , Bovinos , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Interações de Partículas Elementares , Concentração de Íons de Hidrogênio , Luz , Prótons , Reologia/métodos , Espalhamento de Radiação , Soroalbumina Bovina/análise , ViscosidadeRESUMO
PURPOSE: To investigate the underlying cause for the observed differences in self-associating and viscoelastic behavior between two monoclonal antibodies, MAb1, and MAb2. METHODS: Several mutants were designed by swapping charged residues in MAb1 with those present in MAb2 at their respective positions and vice versa. Rheological analysis was done at low and high shear rates. Dynamic light scattering quantified intermolecular interactions in dilute solutions; sedimentation equilibrium analysis determined the corrected weight average molecular weight (M (wc)) to assess the self-associating behavior in high concentration. The molecular charge was estimated from electrophoretic mobility measurements. RESULTS: Replacing the charged residues in the CDR of MAb1 resulted in a lower M (wc) and solution viscosity. The corresponding changes in either just the variable light (VL) or variable heavy (VH) chain showed only a partial decrease in viscosity, whereas changes in both VL and VH chains resulted in a dramatic reduction in viscosity. The converse case where the VL and VH chains of MAb2 were made to look like MAb1 did not self-associate or show increased viscosity. CONCLUSIONS: Exposed charged residues in the CDR of MAb1 are critical in determining the self-associating and highly viscous behavior observed at high concentrations.
Assuntos
Anticorpos Monoclonais/química , Sequência de Aminoácidos , Anticorpos Monoclonais/genética , Dicroísmo Circular , Glicosilação , Humanos , Concentração de Íons de Hidrogênio , Dados de Sequência Molecular , Mutação , Substâncias ViscoelásticasRESUMO
PURPOSE: To explain the differences in protein-protein interactions (PPI) of concentrated versus dilute formulations of a model antibody. METHODS: High frequency rheological measurements from pH 3.0 to 12.0 quantitated viscoelasticity and PPI at high concentrations. Dynamic light scattering (DLS) characterized PPI in dilute solutions. RESULTS: For concentrated solutions at low ionic strength, the storage modulus, a viscosity component and a measure of PPI, is highest at the isoelectric point (pH 9.0) and lowest at pH 5.4. This profile flattens at higher ionic strength but not completely, indicating PPI consist of long-range electrostatics and other short-range attractions. At low concentrations, PPI are near zero at pI but become repulsive as the pH is shifted. Higher salt concentrations completely flatten this profile to zero, indicating that these PPI are mainly electrostatic. CONCLUSIONS: This discrepancy occurs because long-range interactions are significant at low concentrations, whereas both long- and short-range interactions are significant at higher concentrations. Computer modeling was used to calculate antibody properties responsible for long- and short-range interactions, i.e. net charge and dipole moment. Charge-charge interactions are repulsive while dipole-dipole interactions are attractive. Their net effect correlated with the storage modulus profile. However, only charge-charge repulsions correlated with PPI determined by DLS.
Assuntos
Anticorpos/química , Modelos Biológicos , Reologia , Eletricidade Estática , Anticorpos/metabolismo , Dicroísmo Circular , Simulação por Computador , Concentração de Íons de Hidrogênio , Proteínas/metabolismo , Soluções/químicaRESUMO
Feasibility studies were performed to develop a process for obtaining stable dry protein formulations based on in situ polyethylene glycol (PEG)-induced precipitation and vacuum drying of interferon alpha-2a (IFNalpha2a) solution in a vial. Using a laboratory scale freeze dryer, the process was carried out in two phases: first, protein solution containing PEG was concentrated to achieve protein precipitation, and second, remaining water was removed by further reducing the chamber pressure. Drying conditions, i.e. temperature and pressure, and solution composition were selected to ensure maximal precipitation (solubility of IFNalpha2a), to achieve precipitation without boiling, and to ensure stability. Dried formulations were subjected to stability studies (40 degrees C). Concentration and precipitation could be achieved at a fast rate by utilizing pressures slightly above the vapor pressure of water. Fluorescence and circular dichroism (CD) studies showed that precipitated IFNalpha2a maintained its native structure. Fourier transform infrared spectroscopy (FTIR) studies showed that IFNalpha2a when dried in the presence of trehalose, maintained its secondary structure. Trehalose also prevented formation of aggregates during drying. Moisture contents of 1% (w/w) were achieved within 48 h of drying. Dry formulation containing 1:20:100 (w/w) IFNalpha2a:trehalose:mannitol was stable against aggregation and oxidation (6% oxidized at 40 degrees C, 6 months). Stability profile was comparable to a similar lyophilized formulation.
Assuntos
Dessecação/métodos , Interferon-alfa/química , Polietilenoglicóis/química , Precipitação Química , Dicroísmo Circular , Estabilidade de Medicamentos , Armazenamento de Medicamentos , Excipientes/química , Liofilização/métodos , Interferon alfa-2 , Manitol/química , Proteínas Recombinantes , Espectroscopia de Infravermelho com Transformada de Fourier , Temperatura , Trealose/química , VácuoRESUMO
Effect of polyols on the solubility of bovine serum albumin (BSA) in the presence of polyethylene glycols (PEGs) was investigated in order to strengthen the understanding of the observed effects of polyols and PEGs on protein properties in solution. Effect of polyols and/or PEGs on the thermodynamic (conformational) stability of BSA was measured using DSC and circular dichroism (CD). Glucose, sucrose, raffinose, glycerol and sorbitol, all reduced the extent of protein precipitation. Solubility of BSA in the presence of ethylene glycol increased in the case of PEG 1450 and PEG 8000, but was unaffected in the case of PEG 400. DSC studies indicated that smaller PEGs have destabilizing influence on protein structure. CD studies showed that smaller PEGs (ethylene glycol) induce subtle unfolding while stabilizing polyols induce subtle compaction. Results show that, effect of polyols on the apparent solubility of the protein correlates with their effect on the thermodynamic stability of the protein, smaller PEGs are not appropriate for estimating the activity of proteins in saturated solutions, and subtle changes in protein conformation can significantly affect protein precipitation. Though smaller PEGs have weak attractive interactions with protein molecules, perturbation of protein structure by PEGs can be balanced by utilizing appropriate stabilizing solutes.
Assuntos
Polietilenoglicóis/química , Soroalbumina Bovina/química , Álcoois Açúcares/química , Animais , Varredura Diferencial de Calorimetria , Bovinos , Precipitação Química , Dicroísmo Circular , Estabilidade de Medicamentos , Peso Molecular , Estrutura Secundária de Proteína/efeitos dos fármacos , Solubilidade , TermodinâmicaRESUMO
The objective of this work was to investigate the impact of drying method and formulation on the physical stability (aggregation) and selected important physical properties of dried methionyl human growth hormone (Met-hGH) formulations. Solutions of Met-hGH with different stabilizers were dried by different methods (freeze drying, spray drying, and film drying), with and without surfactant. Properties of the dried powders included powder morphology, specific surface area (SSA), protein surface coverage, thermal analysis, and protein secondary structure. Storage stability of Met-hGH in different formulations was also studied at 50 degrees C and at 60 degrees C for 3 months. The dried powders displayed different morphologies, depending mainly on the method of drying and on the presence or absence of surfactant. Film dried powders had the lowest SSA (approximately 0.03 m(2)/g) and the lowest total protein surface accumulation (approximately 0.003%). Surfactant caused a reduction in the SSA of both spray dried and freeze dried powders. Spray dried powders showed greater protein surface coverage and SSA relative to the same formulations dried by other means. Greater in-process perturbations of protein secondary structure were observed with polymer excipients. Formulation impacted physical stability. In general, low molecular weight stabilizers provided better stability. For example, the aggregation rate at 50 degrees C of Met-hGH in a freeze dried trehalose-based formulation was approximately four times smaller than the corresponding Ficoll-70-based formulation. Drying method also influenced physical stability. In general, the film dried preparations studied showed superior stability to preparations dried by other methods, especially those formulations employing low molecular weight stabilizers.