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
The purpose of this work was to elucidate the molecular interactions leading to monoclonal antibody self-association and precipitation and utilize biophysical measurements to predict solubility behavior at high protein concentration. Two monoclonal antibodies (mAb-G and mAb-R) binding to overlapping epitopes were investigated. Precipitation of mAb-G solutions was most prominent at high ionic strength conditions and demonstrated strong dependence on ionic strength, as well as slight dependence on solution pH. At similar conditions no precipitation was observed for mAb-R solutions. Intermolecular interactions (interaction parameter, kD) related well with high concentration solubility behavior of both antibodies. Upon increasing buffer ionic strength, interactions of mAb-R tended to weaken, while those of mAb-G became more attractive. To investigate the role of amino acid sequence on precipitation behavior, mutants were designed by substituting the CDR of mAb-R into the mAb-G framework (GM-1) or deleting two hydrophobic residues in the CDR of mAb-G (GM-2). No precipitation was observed at high ionic strength for either mutant. The molecular interactions of mutants were similar in magnitude to those of mAb-R. The results suggest that presence of hydrophobic groups in the CDR of mAb-G may be responsible for compromising its solubility at high ionic strength conditions since deleting these residues mitigated the solubility issue.
Assuntos
Substituição de Aminoácidos , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Composição de Medicamentos , Imunoglobulina G/química , Mutação Puntual/genética , Sequência de Aminoácidos , Anticorpos Monoclonais/genética , Varredura Diferencial de Calorimetria , Regiões Determinantes de Complementaridade , Humanos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Imunoglobulina G/genética , Concentração Osmolar , Solubilidade , ViscosidadeRESUMO
The objectives of this study were to evaluate the relative binding and potencies of three inhibitors of vascular endothelial growth factor A (VEGF), used to treat neovascular age-related macular degeneration, and assess their relevance in the context of clinical outcome. Ranibizumab is a 48 kDa antigen binding fragment, which lacks a fragment crystallizable (Fc) region and is rapidly cleared from systemic circulation. Aflibercept, a 110 kDa fusion protein, and bevacizumab, a 150 kDa monoclonal antibody, each contain an Fc region. Binding affinities were determined using Biacore analysis. Competitive binding by sedimentation velocity analytical ultracentrifugation (SV-AUC) was used to support the binding affinities determined by Biacore of ranibizumab and aflibercept to VEGF. A bovine retinal microvascular endothelial cell (BREC) proliferation assay was used to measure potency. Biacore measurements were format dependent, especially for aflibercept, suggesting that biologically relevant, true affinities of recombinant VEGF (rhVEGF) and its inhibitors are yet to be determined. Despite this assay format dependency, ranibizumab appeared to be a very tight VEGF binder in all three formats. The results are also very comparable to those reported previously.1-3 At equivalent molar ratios, ranibizumab was able to displace aflibercept from preformed aflibercept/VEGF complexes in solution as assessed by SV-AUC, whereas aflibercept was not able to significantly displace ranibizumab from preformed ranibizumab/VEGF complexes. Ranibizumab, aflibercept, and bevacizumab showed dose-dependent inhibition of BREC proliferation induced by 6 ng/mL VEGF, with average IC50 values of 0.088 ± 0.032, 0.090 ± 0.009, and 0.500 ± 0.091 nM, respectively. Similar results were obtained with 3 ng/mL VEGF. In summary Biacore studies and SV-AUC solution studies show that aflibercept does not bind with higher affinity than ranibizumab to VEGF as recently reported,4 and both inhibitors appeared to be equipotent with respect to their ability to inhibit VEGF function.
Assuntos
Anticorpos Monoclonais Humanizados/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Bevacizumab , Bovinos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Células Endoteliais/citologia , Células Endoteliais/efeitos dos fármacos , Ligação Proteica , Ranibizumab , Ultracentrifugação , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
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
Small-angle neutron scattering (SANS) is used to probe the solution structure of two protein therapeutics (monoclonal antibodies 1 and 2 (MAb1 and MAb2)) and their protein-protein interaction (PPI) at high concentrations. These MAbs differ by small sequence alterations in the complementarity-determining region but show very large differences in solution viscosity. The analyses of SANS patterns as a function of different solution conditions suggest that the average intramolecular structure of both MAbs in solution is not significantly altered over the studied protein concentrations and experimental conditions. Even though a strong repulsive interaction is expected for both MAbs due to their net charges and low solvent ionic strength, analysis of the SANS data shows that the effective PPI for MAb1 is dominated by a very strong attraction at small volume fraction that becomes negligible at large concentrations. The MAb1 PPI cannot be modeled simply by a spherically symmetric central forces model. It is proposed that an anisotropic attraction strongly affects the local interprotein structure and leads to an anomalously large viscosity of concentrated MAb1 solutions. Conversely, MAb2 displays a repulsive interaction potential throughout the concentration series probed and a comparatively small solution viscosity.
Assuntos
Anticorpos Monoclonais Humanizados/química , Animais , Anisotropia , Anticorpos Monoclonais Humanizados/metabolismo , Células CHO , Cricetinae , Cricetulus , Humanos , Difração de Nêutrons , Concentração Osmolar , Ligação Proteica , Conformação Proteica , Espalhamento a Baixo Ângulo , Soluções/química , ViscosidadeRESUMO
Weak protein-protein interactions are thought to modulate the viscoelastic properties of concentrated antibody solutions. Predicting the viscoelastic behavior of concentrated antibodies from their dilute solution behavior is of significant interest and remains a challenge. Here, we show that the diffusion interaction parameter (k(D)), a component of the osmotic second virial coefficient (B(2)) that is amenable to high-throughput measurement in dilute solutions, correlates well with the viscosity of concentrated monoclonal antibody (mAb) solutions. We measured the k(D) of 29 different mAbs (IgG(1) and IgG(4)) in four different solvent conditions (low and high ion normality) and found a linear dependence between k(D) and the exponential coefficient that describes the viscosity concentration profiles (|R| ≥ 0.9). Through experimentally measured effective charge measurements, under low ion normality where the electroviscous effect can dominate, we show that the mAb solution viscosity is poorly correlated with the mAb net charge (|R| ≤ 0.6). With this large data set, our results provide compelling evidence in support of weak intermolecular interactions, in contrast to the notion that the electroviscous effect is important in governing the viscoelastic behavior of concentrated mAb solutions. Our approach is particularly applicable as a screening tool for selecting mAbs with desirable viscosity properties early during lead candidate selection.
Assuntos
Anticorpos Monoclonais/química , Ensaios de Triagem em Larga Escala , Animais , Células CHO , Cricetinae , Cricetulus , Difusão , Concentração Osmolar , Solventes , ViscosidadeRESUMO
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
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 investigate the cause of the observed instability of dulanermin in 100 ml polyolefin (PO) infusion bags containing saline. METHODS: Diluted dulanermin in IV bags was collected and frozen prior to analysis by size exclusion chromatography. The UV absorption profiles of the IV bag solutions were characterized by using spectrophotometry. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) measured the metal content. Leachables from IV bags were identified by LC-UV-high resolution MS/MS analysis. RESULTS: An elevated loss of dulanermin monomers was observed only in 100 ml PO bags. These IV bag solutions have a compound that contains zinc and has absorbance at 320 nm. This compound was identified to be 2-Mercaptobenzothiazole, and its zinc salt and was found to come from the stopper used in the 100 ml PO bags. The manufacturer has subsequently corrected this problem by using non-latex components in the 100 ml PO IV bag. CONCLUSIONS: End-users need to be aware that IV bags made from a particular polymer by the same manufacturer may contain components or use a manufacturing process that results in a different product. Analysis of samples after freezing and thawing proved to be useful in identifying potential incompatibility of dulanermin in the IV bags.
Assuntos
Infusões Intravenosas , Proteínas/administração & dosagem , Cloreto de Sódio , Cromatografia Líquida de Alta Pressão , Espectrometria de Massas/métodos , Espectrofotometria UltravioletaRESUMO
PURPOSE: To study recombinant human vascular endothelial growth factor (rhVEGF), the release characteristics from topical gel formulations, and its interaction with the gelling agents. METHODS: The release kinetics were followed by quantifying rhVEGF that diffused into the receptor chamber of Franz cells. Analytical ultracentrifuge (AUC) was used to characterize the sedimentation velocity of rhVEGF experienced in the gel. The interactions were characterized by isothermal calorimetry (ITC), and rhVEGF conformation was assessed by circular dichroism (CD). RESULTS: The fraction of protein released was linear with the square root of time. The release rate constants did not show significant change within a wide range of bulk viscosities created by different concentrations of hydroxypropyl methylcellulose (HPMC) or MC gels. Sedimentation velocity determined by AUC generated comparable sedimentation coefficients of protein in these gels. AUC and ITC revealed no significant interaction between rhVEGF and HPMC and some change on secondary structure of the protein by Far UV CD, which was not the case with carboxymethyl cellulose (CMC). CONCLUSIONS: Microviscosity, not bulk viscosity, was the key factor for the release of rhVEGF from cellulosic gels such as HPMC. Interaction between rhVEGF and CMC resulted in slower, and reduced amount of, release from the gel.
Assuntos
Sistemas de Liberação de Medicamentos , Géis/química , Fator A de Crescimento do Endotélio Vascular/administração & dosagem , Administração Tópica , Dicroísmo Circular , Difusão , Humanos , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Ultracentrifugação , Fator A de Crescimento do Endotélio Vascular/química , ViscosidadeRESUMO
Noncovalent molecular interactions, which are central to life, are thermodynamic processes that follow common interaction pathways. This commentary provides a foundation for both considering noncovalent interactions and the interplay between the protein properties and the solvent properties in determining the energetics. In biopharmaceutics, noncovalent interactions are a 2-edged sword. Foremost, they provide a core function for biopharmaceutical agents, binding to targets, substrates, or receptors. At the same time, they are at the root of the solubility and viscosity difficulties encountered in the manufacture, formulation, and delivery of protein-based pharmaceuticals. This commentary describes the interaction process and summarizes the energetics of the interaction pathway. The focus will be on protein-protein interactions, while recognizing that the processes and energetics are entirely general and applicable to all solution interactions. The contributions of protein molecular properties and protein colloidal properties to the pathway are described, and the relationship between the two is developed. The processes leading to protein-protein binding are described with respect to the attractive interactions that lead to aggregation and high viscosity. The concept of emergent heterogeneity is introduced, and a model presented for how noncontacting interactions may lead to high viscosities without simultaneously causing low solubility.
Assuntos
Biofarmácia/métodos , Coloides/metabolismo , Domínios e Motivos de Interação entre Proteínas/fisiologia , Coloides/química , Ligação Proteica/fisiologia , Solubilidade , ViscosidadeRESUMO
During the development of protein therapeutics, characterization of the active pharmaceutical ingredient is performed extensively to ensure the stability, safety, and efficacy of the drug. Little is known, however, about the characteristics of protein drugs circulating in the blood. The recent availability of a fluorescence detection system (FDS) in analytical ultracentrifugation (AUC) instruments enables the characterization of fluorescently labeled proteins in biological fluids. AUC provides information about protein size, shape, self-association, and binding while avoiding many limitations associated with size exclusion chromatography. Furthermore, with the specificity and sensitivity of FDS, measurements can be performed at physiological concentrations directly in serum. In the current study, we used omalizumab, an anti-immunoglobulin E (IgE) monoclonal antibody, to demonstrate the potential of using AUC-FDS for the study of a monoclonal antibody and its complexes directly in human serum. Omalizumab properties were essentially unaltered after labeling with the fluorescent dye Alexa Fluor 488. In addition, omalizumab and IgE formed different complexes in serum than in phosphate-buffered saline in terms of both size and affinity.
Assuntos
Anticorpos Monoclonais/sangue , Anticorpos Monoclonais/metabolismo , Complexo Antígeno-Anticorpo/sangue , Imunoglobulina E/sangue , Imunoglobulina E/metabolismo , Soro , Ultracentrifugação/métodos , Animais , Anticorpos Anti-Idiotípicos , Anticorpos Monoclonais Humanizados , Células CHO , Linhagem Celular Tumoral , Cromatografia em Gel , Cricetinae , Cricetulus , Ensaio de Imunoadsorção Enzimática , Humanos , OmalizumabRESUMO
Multiple molecular driving forces mediate protein stability, association, and recognition in concentrated solutions. Here we investigate the interactions that modulate the nonideal solution behavior of two immunoglobulins (IgG1s) in highly concentrated solutions using two-dimensional vibrational correlation spectroscopy (2D-COS) and principal components analysis (PCA). A specific sequence of changes is observed in the concentration-dependent vibrational spectra of the highly viscous IgG solution that deviates from ideality, whereas that sequence is reversed for all other conditions examined. The asynchronous spectra reveal variation in beta-sheet and turn regions occur before intensity variations in disordered and alpha-helical regions as the concentration is increased for the highly viscous regime. This is in contrast to the sequence observed for all other conditions studied and to the idea that beta-sheet regions are resistant to concentration-dependent affects. Finally, we show that increased hydrogen bonding and electrostatics primarily modulate the intermolecular association and nonideal behavior. Specifically, 2D-COS and PCA analysis of the amide II region suggests that Glu and Asp residues trigger the change resulting in increased viscosity and association of one IgG.
Assuntos
Imunoglobulina G/análise , Imunoglobulina G/química , Ligação de Hidrogênio , Análise de Componente Principal , Conformação Proteica , Soluções , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade Estática , ViscosidadeRESUMO
PURPOSE: This paper shows the first ever assembly of monoclonal antibody using multivalent carboxylate ions into highly ordered structures that feature viscoelastic properties reminiscent of other filamentous proteins. METHODS: A monoclonal antibody was assembled into filamentous networks by adding multivalent carboxylates to the protein solution. Gelation and characterization of these networks were monitored using mechanical rheometry, electron microscopy, Fourier transform infra-red and Raman spectroscopy. RESULTS: Electron microscopy and mechanical rheometry suggest the formation of rigid filament bundles that feature strong interfilament interactions. Filament network elasticity increased with multivalent carboxylate and protein concentrations, hinting at the importance of multivalent carboxylates in the mechanism of assembly. CONCLUSION: Assembly is not triggered by high ionic strength but with multivalent carboxylates. A high protein concentration is required for filament formation and the elasticity of the networks are weakly dependent on concentration. The exact mechanism of assembly is still elusive, although we speculate that carboxylates could act as a bridge to crosslink antibody monomers. These monoclonal antibody monomers could be linked either through Fab-Fab or Fc-Fab regions, although previous reports have shown evidence of reversible self-association mediated through the Fab regions.
Assuntos
Anticorpos Monoclonais/química , Ácidos Carboxílicos/química , Géis/química , Anticorpos Monoclonais/ultraestrutura , Citratos/química , Elasticidade , Humanos , Espectroscopia de Infravermelho com Transformada de Fourier , ViscosidadeRESUMO
The aspartate residue (Asp 32) located in the complementarity-determining region (CDR) of a recombinant humanized monoclonal antibody (MAb I) is highly susceptible to the isomerization reaction. The modification of Asp 32 residue due to the isomerization reaction results in a significant reduction in the binding affinity of MAb I to IgE. The binding of a MAb I therapeutic to IgE is important for its desired pharmacological effect. In earlier investigations, we demonstrated that the conformational flexibility and residue exposure are factors that are responsible for the observed reactivity of Asp 32 in MAb I. This report explores the role of cosolutes such as glycerol and sucrose in the modulation of Asp 32 reactivity in MAb I. These cosolutes are routinely incorporated in injectable pharmaceutical formulations. The reactivity of the Asp residue in MAb I in these different cosolute-based formulations was compared to its reactivity in a peptide model VDYDG comprising residues 29-33 of MAb I. The formulations of MAb I and VDYDG containing varying concentrations of glycerol and sucrose were incubated at 50 degrees C for a period of 5-7 days. The isomerization of the Asp residue in VDYDG and MAb I was monitored using rp-HPLC and hydrophobic interaction chromatography (HIC), respectively. Structural analysis of MAb I using differential scanning calorimetry (DSC) demonstrated that the structural stability of MAb I was increased in formulations containing glycerol and sucrose. However, the stability of Asp 32 in MAb I was significantly decreased in these formulations. This research suggests that a formulation approach that relies purely on enhancing the structural stability of proteins through addition of these cosolutes could result in problems associated with the chemical stability of these biomolecules.
Assuntos
Anticorpos Monoclonais/química , Ácido Aspártico/química , Excipientes/química , Glicerol/química , Sacarose/química , Anticorpos Monoclonais/metabolismo , Afinidade de Anticorpos , Varredura Diferencial de Calorimetria , Química Farmacêutica , Estabilidade de Medicamentos , Concentração de Íons de Hidrogênio , Imunoglobulina E/metabolismo , Isomerismo , Cloreto de Magnésio/química , Espectroscopia de Ressonância Magnética , Modelos Químicos , Oligopeptídeos , Conformação Proteica , Desnaturação Proteica , Proteínas Recombinantes/química , Cloreto de Sódio/química , Espectrometria de Fluorescência , Tecnologia Farmacêutica/métodos , TemperaturaRESUMO
This study was conducted to investigate the effect of reversible protein self-association on the viscosity of concentrated monoclonal antibody solutions. The viscosities of the monoclonal antibody solutions were measured by either a capillary viscometer or a cone-plate rheometer at different protein concentrations, pH, and ionic strength. Soluble aggregates were determined by size exclusion chromatography, light scattering, and analytical ultracentrifugation. Self-association of protein at high protein concentration was monitored by sedimentation equilibrium analysis using a preparative ultracentrifuge and a microfractionator. The viscosity of one of the monoclonal antibodies investigated is highly dependent on protein concentration, pH, and ionic strength of buffer and charged excipients. This antibody shows the highest viscosity near its pI at low ionic strength conditions. Sedimentation equilibrium analysis suggests that this antibody tends to reversibly self-associate at high protein concentration. The self-association appears to be quite weak and is not detectable by sedimentation velocity and size exclusion chromatography at low protein concentration. There are no significant differences in the amounts of non-dissociable soluble aggregates formed between low viscosity and high viscosity samples. These results suggest that the reversible multivalent self-association of this protein appears to be mediated mainly by electrostatic interactions of charged residues and results in unusually high viscosity of this monoclonal antibody in solution at low ionic strength conditions.