RESUMEN
Different factors affect the long term stability of monoclonal antibodies, among them denaturation or partial denaturation that is often followed by aggregation. Isothermal calorimetry is capable of quantifying the kinetics of denaturation/aggregation of an antibody by measuring the heat that is released or absorbed by the process over a period of days or weeks, at temperatures below its denaturation temperature, Tm. The denaturation/aggregation kinetics of the anti-HIV monoclonal antibody VRC07-523LS was measured by isothermal calorimetry at different concentrations in four different formulation buffers. The measurements were performed at ten degrees below Tm, as determined by differential scanning calorimetry. The formation of aggregates was also followed by size exclusion chromatography at 5⯰C, 25⯰C and 40⯰C over a period of 8-36 weeks. It was observed that the rates measured by isothermal calorimetry correlate quantitatively with those measured by size exclusion chromatography. Since isothermal calorimetry experiments are performed over a period of ten days, it can become a valuable tool for a fast prediction of the best formulations.
Asunto(s)
Anticuerpos Anti-VIH/química , VIH-1/inmunología , Anticuerpos Monoclonales/química , Anticuerpos Neutralizantes/química , Apraxia Ideomotora , Calorimetría/métodos , Rastreo Diferencial de Calorimetría/métodos , Calor , Humanos , Agregado de Proteínas , Desnaturalización Proteica , Estabilidad ProteicaRESUMEN
Structure-based vaccine design has been used to develop immunogens that display conserved neutralization sites on pathogens such as HIV-1, respiratory syncytial virus (RSV), and influenza. Improving the immunogenicity of these designed immunogens with adjuvants will require formulations that do not alter protein antigenicity. Here, we show that nanoparticle-forming thermoresponsive polymers (TRP) allow for co-delivery of RSV fusion (F) protein trimers with Toll-like receptor 7 and 8 agonists (TLR-7/8a) to enhance protective immunity. Although primary amine conjugation of TLR-7/8a to F trimers severely disrupted the recognition of critical neutralizing epitopes, F trimers site-selectively coupled to TRP nanoparticles retained appropriate antigenicity and elicited high titers of prefusion-specific, TH1 isotype anti-RSV F antibodies following vaccination. Moreover, coupling F trimers to TRP delivering TLR-7/8a resulted in â¼3-fold higher binding and neutralizing antibody titers than soluble F trimers admixed with TLR-7/8a and conferred protection from intranasal RSV challenge. Overall, these data show that TRP nanoparticles may provide a broadly applicable platform for eliciting neutralizing antibodies to structure-dependent epitopes on RSV, influenza, HIV-1, or other pathogens.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Nanopartículas/administración & dosificación , Polímeros/química , Vacunas contra Virus Sincitial Respiratorio/farmacología , Proteínas Virales de Fusión/administración & dosificación , Animales , Anticuerpos Neutralizantes , Técnicas de Química Sintética , Sistemas de Liberación de Medicamentos/métodos , Femenino , Ratones Endogámicos , Nanopartículas/química , Vacunas contra Virus Sincitial Respiratorio/administración & dosificación , Vacunas contra Virus Sincitial Respiratorio/inmunología , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 8/agonistas , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/química , Proteínas Virales de Fusión/químicaRESUMEN
Broadly neutralizing antibodies are showing promise in the treatment and prevention of HIV-1, with several now being evaluated clinically. Some lead clinical candidates, including antibodies CAP256-VRC26.25, N6, PGT121, and VRC07-523, have one or more N-linked glycosylation sequons in their variable domains (Fvs) from somatic hypermutation, and these glycans increase chemical heterogeneity, complicating the manufacture of these antibodies as products. Here we propose a general method to remove Fv glycans and use this method to develop engineered versions of these four antibodies with Fv glycans removed. When germline residues were introduced to remove each glycan, antibody properties between wild type and mutant were not significantly altered for CAP256-VRC26.25 and PGT121; however, germline mutants for N6 and VRC07-523 showed increased polyreactivity, which is known to correlate with unfavorable in vivo pharmacokinetics. To reduce polyreactivity induced by removal of Fv glycan, we mutated aromatic residues and arginines structurally proximal to the removed glycan and identified Fv glycan-removed variants with low polyreactivity for N6 and VRC07-523. Two such variants, N6-N72LCQ-R18LCD and VRC07-523-N72LCQ-R24LCD, showed thermostability, neutralization potency and breadth, and half-life in humanized FcRn mice that were similar to their wild-type Fv-glycosylated counterparts. The removal of Fv glycan and reduction of chemical heterogeneity were confirmed by liquid chromatography-mass spectrometry. With reduced heterogeneity, the Fv-glycan-removed variants developed here may have utility as products for treating or preventing infection by HIV-1.
Asunto(s)
Anticuerpos ampliamente neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1 , Región Variable de Inmunoglobulina/inmunología , Animales , Glicosilación , Anticuerpos Anti-VIH/química , Infecciones por VIH/prevención & control , Humanos , Región Variable de Inmunoglobulina/química , Ratones , PolisacáridosRESUMEN
Highly effective HIV-1-neutralizing antibodies could have utility in the prevention or treatment of HIV-1 infection. To improve the potency of 10E8, an antibody capable of near pan-HIV-1 neutralization, we engineered 10E8-surface mutants and screened for improved neutralization. Variants with the largest functional enhancements involved the addition of hydrophobic or positively charged residues, which were positioned to interact with viral membrane lipids or viral glycan-sialic acids, respectively. In both cases, the site of improvement was spatially separated from the region of antibody mediating molecular contact with the protein component of the antigen, thereby improving peripheral semi-specific interactions while maintaining unmodified dominant contacts responsible for broad recognition. The optimized 10E8 antibody, with mutations to phenylalanine and arginine, retained the extraordinary breadth of 10E8 but with â¼10-fold increased potency. We propose surface-matrix screening as a general method to improve antibodies, with improved semi-specific interactions between antibody and antigen enabling increased potency without compromising breadth.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Membrana Celular/metabolismo , Proteína gp41 de Envoltorio del VIH/metabolismo , Semivida , Humanos , Pruebas de Neutralización , Polisacáridos/metabolismo , Unión ProteicaRESUMEN
This study describes the physicochemical characterization, stabilization, and formulation design of SM4-AC, an acrylodan-labeled glucose/galactose-binding protein for use in a continuous glucose monitoring device. The physical stability profile of SM4-AC as a function of pH and temperature was monitored using a combination of biophysical techniques and the resulting physical stability profile was visualized using an empirical phase diagram. Forced degradation chemical stability studies (Asn deamidation, Met oxidation) of SM4-AC were performed using a combination of capillary isoelectric focusing, peptide mapping, and reversed-phase HPLC. Differential scanning fluorimetry was then employed to screen various pharmaceutical excipients for their ability to physically stabilize SM4-AC. An optimized formulation of 20% sucrose and 2.5 mM calcium chloride in 10 mM MES buffer, 150 mM NaCl at pH 6.0 increased the conformational stability of SM4-AC by 15°C. Accelerated and real-time stability studies were setup to compare the SM4-AC protein's physicochemical stability and glucose-binding activity in 2 formulations for up to 12 months. SM4-AC in an optimized formulation (vs the original formulation) showed improved physical stability, and similar chemical stability and glucose binding activity profiles during storage up to 52 weeks at various temperatures.
Asunto(s)
2-Naftilamina/análogos & derivados , Química Farmacéutica/métodos , Composición de Medicamentos/métodos , Diseño de Fármacos , Glucosa/química , Glucosa/metabolismo , 2-Naftilamina/química , 2-Naftilamina/metabolismo , Dicroismo Circular/métodos , Estabilidad de Medicamentos , Unión Proteica/fisiologíaRESUMEN
Continuous glucose monitoring (CGM) devices offer diabetes patients a convenient approach to assist in controlling blood glucose levels. A prototype CGM has been developed that uses the emission profile of a polarity-sensitive fluorophore (acrylodan) conjugated to a glucose/galactose-binding protein (SM4-AC) to measure the concentration of glucose in vivo. During development, a decrease in the devices signal intensity was observed in vivo over time, which was postulated to be result of oxidative degradation of SM4-AC. A comprehensive physicochemical analysis of SM4-AC was pursued to identify potential mechanisms of signal intensity loss in this CGM during in vitro forced oxidation studies. An assessment of the structural integrity and conformational stability of SM4-AC indicated a relatively decreased polarity and lower tertiary structure stability compared to unconjugated protein (SM4). The stability and polarity of SM4-AC was also altered in the presence of H2 O2 . Furthermore, a time-dependent loss in the fluorescence signal of SM4-AC was observed when incubated with H2 O2 . An LC-MS peptide mapping analysis of these protein samples indicated that primarily two Met residues in SM4-AC were susceptible to oxidation. When these two residues were genetically altered to an amino acid not prone to oxidation, the glucose binding ability of the protein was retained and no loss of acrylodan fluorescence was observed in the presence of H2 O2 . Genetic alteration of these two residues is proposed as an effective approach to increase the long-term stability of SM4-AC within this prototype CGM in vivo.
Asunto(s)
2-Naftilamina/análogos & derivados , Proteínas de Escherichia coli/química , Escherichia coli/química , 2-Naftilamina/química , Automonitorización de la Glucosa Sanguínea/métodos , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Oxidación-Reducción , Dominios Proteicos , Estabilidad ProteicaRESUMEN
Stabilization and formulation of therapeutic proteins against physical instability, both structural alterations and aggregation, is particularly challenging not only due to each protein's unique physicochemical characteristics but also their susceptibility to the surrounding milieu (pH, ionic strength, excipients, etc.) as well as various environmental stresses (temperature, agitation, lyophilization, etc.). The use of high-throughput techniques can significantly aid in the evaluation of stabilizing solution conditions by permitting a more rapid evaluation of a large matrix of possible combinations. In this mini-review, we discuss both key physical degradation pathways observed for protein-based drugs and the utility of various high-throughput biophysical techniques to aid in protein formulation development to minimize their occurrence. We then focus on four illustrative case studies with therapeutic protein candidates of varying sizes, shapes and physicochemical properties to explore different analytical challenges in monitoring protein physical instability. These include an IgG2 monoclonal antibody, an albumin-fusion protein, a recombinant pentameric plasma glycoprotein, and an antibody fragment (Fab). Future challenges and opportunities to improve and apply high-throughput approaches to protein formulation development are also discussed.