RESUMEN
The presence of protein aggregates is commonly believed to be an important risk factor for immunogenicity of therapeutic proteins. Among all types of aggregates, dimers are relatively abundant in most commercialized monoclonal antibody (mAb) products. The aim of this study was to investigate the immunogenicity of artificially created mAb dimers relative to that of unstressed and stressed mAb monomers. A monoclonal murine IgG1 (mIgG1) antibody was exposed to low pH, elevated temperature, or UV irradiation to induce dimerization. Dimers and monomers were purified via size-exclusion chromatography. Physicochemical analysis revealed that upon all stress conditions, new deamidation or oxidation or both of amino acids occurred. Nevertheless, the secondary and tertiary structures of all obtained dimers were similar to those of unstressed mIgG1. Isolated dimers were administered subcutaneously in Balb/c mice, and development of antidrug antibodies and accumulation of follicular T helper cells in draining lymph nodes and spleens were determined. None of the tested dimers or stressed monomers were found to be more immunogenic than the unstressed control in our mouse model. In conclusion, both dimers and monomers generated by using 3 different stress factors have a low immunogenicity similar to that of the unstressed monomers.
Asunto(s)
Anticuerpos Monoclonales/administración & dosificación , Animales , Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Composición de Medicamentos , Estabilidad de Medicamentos , Concentración de Iones de Hidrógeno , Inyecciones Subcutáneas , Ganglios Linfáticos/efectos de los fármacos , Ganglios Linfáticos/inmunología , Ratones Endogámicos BALB C , Oxidación-Reducción , Agregado de Proteínas , Multimerización de Proteína , Estabilidad Proteica , Bazo/efectos de los fármacos , Bazo/inmunología , Linfocitos T Colaboradores-Inductores/efectos de los fármacos , Linfocitos T Colaboradores-Inductores/inmunología , Temperatura , Rayos UltravioletaRESUMEN
Protein aggregates are one of the several risk factors for undesired immunogenicity of biopharmaceuticals. However, it remains unclear which features determine whether aggregates will trigger an unwanted immune response. The aim of this study was to determine the effect of aggregates' size on their relative immunogenicity. A monoclonal murine IgG1 was stressed by exposure to low pH and elevated temperature followed by stirring to obtain aggregates widely differing in size. Aggregate fractions enriched in soluble oligomers, submicron size particles and micron size particles were isolated via centrifugation or size-exclusion chromatography and characterized physicochemically. The secondary and tertiary structures of aggregates were altered in a similar way for all the fractions, while no substantial chemical degradation was observed. Development of anti-drug antibodies was measured after subcutaneous administration of each enriched fraction to BALB/c mice. Among all tested fractions, the most immunogenic was the one highly enriched in submicron size particles (â¼100-1000 nm). Fractions composed of micron size (>1-100 µm) particles or soluble oligomers (<100 nm) were not immunogenic under the dosing regimen studied in this work. These results show that aggregate size is an important factor for protein immunogenicity.
Asunto(s)
Anticuerpos Monoclonales/química , Anticuerpos Monoclonales/inmunología , Hipersensibilidad a las Drogas/inmunología , Inmunoglobulina G/química , Inmunoglobulina G/inmunología , Agregado de Proteínas , Animales , Anticuerpos Monoclonales/efectos adversos , Formación de Anticuerpos , Inmunoglobulina G/efectos adversos , Ratones Endogámicos BALB C , Tamaño de la PartículaRESUMEN
Antigenic drift of the influenza A virus requires that vaccine production is targeted to the strains circulating each year. Live-attenuated influenza A vaccine manufacturing is used to produce intact virions with the surface antigens of the circulating strains. Influenza A typically contains a large percentage (>90%) of non-infective virions. The ribonucleoprotein (RNP) content, virion structure, and aggregation are factors that are thought to have an impact on infectivity. However, these factors are difficult to study because of the intrinsic variability in virion size, shape and overall structural integrity. Negative stain TEM for total particle counts and cryoTEM for detailed size/structural analysis are established benchmark techniques for virus characterization. Other methods may be valuable for certain sample types or circumstances. The aim of this work is to establish a benchmark comparison between orthogonal biophysical techniques for particle counts, population size distribution, structural integrity, and aggregate levels. NTA and FFF-MALS rapidly provided total counts, size distribution, and aggregate/elongated virion content. CryoTEM with size analysis and fraction counting yielded detailed information about the pleomorphism of the sample. The structural integrity of virions was inferred from multi-signal AUC-SV and CryoTEM. The current work provides a comparative assessment and a baseline for the selection of biophysical tools for the determination of particle counts, aggregation and pleomorphic characteristics of influenza A virus.