RESUMEN
Excipients are added to biopharmaceutical formulations to enhance protein stability and enable the development of robust formulations with acceptable physicochemical properties, but the mechanism by which they confer stability is not fully understood. Here, we aimed to elucidate the mechanism through direct experimental evidence of the binding affinity of an excipient to a monoclonal antibody (mAb), using saturation transfer difference (STD) nuclear magnetic resonance (NMR) spectroscopic method. We ranked a series of excipients with respect to their dissociation constant (KD) and nonspecific binding constants (Ns). In parallel, molecular dynamic and site identification by ligand competitive saturation (SILCS)-Monte Carlo simulations were done to rank the excipient proximity to the proteins, thereby corroborating the ranking by STD NMR. Finally, the excipient ranking by NMR was correlated with mAb conformational and colloidal stability. Our approach can aid excipient selection in biologic formulations by providing insights into mAb-excipient affinities before conventional and time-consuming excipient screening studies are conducted.
Asunto(s)
Productos Biológicos , Excipientes , Anticuerpos Monoclonales/química , Espectroscopía de Resonancia Magnética/métodos , Conformación MolecularRESUMEN
Subcutaneous injection of a low volume (<2 mL) high concentration (>100 mg/mL) formulation is an attractive administration strategy for monoclonal antibodies (mAbs) and other biopharmaceutical proteins. Using concentrated solutions may also be beneficial at various stages of bioprocessing. However, concentrating proteins by conventional techniques, such as ultrafiltration, can be time consuming and challenging. Isolation of the dense fraction produced by macroscopic liquid-liquid phase separation (LLPS) has been suggested as a means to produce high-concentration solutions, but practicality of this method, and the stability of the resulting protein solution have not previously been demonstrated. In this proof-of-concept study, we demonstrate that LLPS can be used to concentrate a mAb solution to >170 mg/mL. We show that the structure of the mAb is not altered by LLPS, and unperturbed mAb is recoverable following dilution of the dense fraction, as judged by 1H nuclear magnetic resonance spectroscopy. Finally, we show that the physical properties and stability of a model high concentration protein formulation obtained from the dense fraction can be improved, for example through the addition of the excipient arginine·glutamate. This results in a stable high-concentration protein formulation with reduced viscosity and no further macroscopic LLPS. Concentrating mAb solutions by LLPS represents a simple and effective technique to progress toward producing high-concentration protein formulations for bioprocessing or administration.AbbreviationsArginine·glutamate (Arg·Glu), Carr-Purcell-Meiboom-Gill (CPMG), critical temperature (TC), high-performance size-exclusion chromatography (HPSEC), liquid-liquid phase separation (LLPS), monoclonal antibody (mAb), nuclear magnetic resonance (NMR), transverse relaxation rate (R2).
Asunto(s)
Anticuerpos Monoclonales/química , Extracción Líquido-Líquido/métodos , Estabilidad Proteica , Química Farmacéutica/métodos , Humanos , Soluciones Farmacéuticas/química , Prueba de Estudio ConceptualRESUMEN
PURPOSE: To investigate differences in the preferential exclusion of trehalose, sucrose, sorbitol and mannitol from the surface of three IgG1 monoclonal antibodies (mAbs) and understand its effect on the aggregation and reversible self-association of mAbs at high-concentrations. METHODS: Preferential exclusion was measured using vapor pressure osmometry. Effect of excipient addition on accelerated aggregation kinetics was quantified using size exclusion chromatography and on reversible self-association was quantified using dynamic light scattering. RESULTS: The doubling of excipient concentration in the 0 to 0.5 m range resulted in a doubling of the mAb transfer free energy for all excipients and antibodies tested in this study. Solution pH and choice of buffering agent did not significantly affect the magnitude of preferential exclusion. We find that aggregation suppression for trehalose, sucrose and sorbitol (but not mannitol) correlates with the magnitude of their preferential exclusion from the native state of the three IgG1 mAbs. We also find that addition of sugars and polyols reduced the tendency for reversible self-association in two mAbs that had weakly repulsive or neutral self-interactions in the presence of buffer alone. CONCLUSIONS: The magnitude of preferential exclusion for trehalose, sucrose and sorbitol correlates well with their partial molar volumes in solution. Mannitol is excluded to a greater extent than that expected from its partial molar volume, suggesting specific interactions of mannitol that might be different than the other sugars and polyols tested in this study. Local interactions play a role in the effect of excipient addition on the reversible self-association of mAbs. These results provide further insights into the stabilization of high-concentration mAb formulations by sugars and polyols.
Asunto(s)
Anticuerpos Monoclonales/química , Inmunoglobulina G/química , Polímeros/química , Agregado de Proteínas , Sacarosa/química , Alcoholes del Azúcar/química , Trehalosa/química , Excipientes/química , Cinética , Simulación de Dinámica Molecular , Conformación Proteica , Propiedades de SuperficieRESUMEN
Preferential interactions of weakly interacting formulation excipients govern their effect on the equilibrium and kinetics of several reactions of protein molecules in solution. Using vapor pressure osmometry, we characterized the preferential interactions of commonly used excipients trehalose, L-arginine.HCl and NaCl with three therapeutically-relevant, IgG1 monoclonal antibodies that have similar size and shape, but differ in their surface hydrophobicity and net charge. We further characterized the effect of these excipients on the reversible self-association, aggregation and viscosity behavior of these antibody molecules. We report that trehalose, L-arginine.HCl and NaCl are all excluded from the surface of the three IgG1 monoclonal antibodies, and that the exclusion behavior is linearly related to the excipient molality in the case of trehalose and NaCl, whereas a non-linear behavior is observed for L-arginine.HCl. Interestingly, we find that the magnitude of trehalose exclusion depends upon the nature of the protein surface. Such behavior is not observed in case of NaCl and L-arginine.HCl as they are excluded to the same extent from the surface of all three antibody molecules tested in this study. Analysis of data presented in this study provides further insight into the mechanisms governing excipient-mediated stabilization of mAb formulations.
Asunto(s)
Anticuerpos Monoclonales/efectos de los fármacos , Arginina/farmacología , Inmunoglobulina G/efectos de los fármacos , Cloruro de Sodio/farmacología , Trehalosa/farmacología , Estabilidad de Medicamentos , Excipientes/farmacología , OsmometriaRESUMEN
Assessing how excipients affect the self-association of monoclonal antibodies (mAbs) requires informative and direct in situ measurements for highly concentrated solutions, without sample dilution or perturbation. This study explores the application of solution nuclear magnetic resonance (NMR) spectroscopy for characterization of typical mAb behavior in formulations containing arginine glutamate. The data show that the analysis of signal intensities in 1D 1H NMR spectra, when compensated for changes in buffer viscosity, is invaluable for identifying conditions where protein-protein interactions are minimized. NMR-derived molecular translational diffusion rates for concentrated solutions are less useful than transverse relaxation rates as parameters defining optimal formulation. Furthermore, NMR reports on the solution viscosity and mAb aggregation during accelerated stability study assessment, generating data consistent with that acquired by size-exclusion chromatography. The methodology developed here offers NMR spectroscopy as a new tool providing complementary information useful to formulation development of mAbs and other large therapeutic proteins.
Asunto(s)
Anticuerpos Monoclonales/análisis , Dipéptidos/análisis , Excipientes/análisis , Espectroscopía de Resonancia Magnética/métodos , Anticuerpos Monoclonales/química , Química Farmacéutica/métodos , Dipéptidos/química , Estabilidad de Medicamentos , Excipientes/química , Humanos , Soluciones Farmacéuticas/análisis , Soluciones Farmacéuticas/químicaRESUMEN
Monoclonal antibodies display complicated solution properties in highly concentrated (>100 mg/mL) formulations, such as high viscosity, high aggregation propensity, and low stability, among others, originating from protein-protein interactions within the colloidal protein solution. These properties severely hinder the successful development of high-concentration mAb solution for subcutaneous injection. We hereby investigated the effects of several small-molecule excipients with diverse biophysical-chemical properties on the viscosity, aggregation propensity, and stability on two model IgG1 (JM1 and JM2) mAb formulations. These excipients include nine amino acids or their salt forms (Ala, Pro, Val, Gly, Ser, HisHCl, LysHCl, ArgHCl, and NaGlu), four representative salts (NaCl, NaAc, Na2SO4, and NH4Cl), and two chaotropic reagents (urea and GdnHCl). With only salts or amino acids in their salt-forms, significant decrease in viscosity was observed for JM1 (by up to 30-40%) and JM2 (by up to 50-80%) formulations, suggesting charge-charge interaction between the mAbs dictates the high viscosity of these mAbs formulations. Most of these viscosity-lowering excipients did not induce substantial protein aggregation or changes in the secondary structure of the mAbs, as evidenced by HPLC-SEC, DSC, and FT-IR analysis, even in the absence of common protein stabilizers such as sugars and surfactants. Therefore, amino acids in their salt-forms and several common salts, such as ArgHCl, HisHCl, LysHCl, NaCl, Na2SO4, and NaAc, could potentially serve as viscosity-lowering excipients during high-concentration mAb formulation development.