RESUMO
The lack of a fast selection method to identify the most stable protein is one of the major challenges for developing successful therapeutic protein formulations more rapidly. The swift and accurate detection of small amounts of aggregates is another problem since aggregates may trigger an immunological response and the aggregation decreases the biological activity of the antibody. Here we present an alternative method for initial screening of the aggregation propensity of proteins, using monoclonal antibodies (mAb) as an example and thioflavin T (ThT) binding. The major advantage of ThT binding is the short duration of testing compared with size-exclusion chromatography (SEC) measurements that can take 6 months or more even under accelerated conditions. The tendency to aggregate of each therapeutic human mAb probed with the ThT assay, together with SEC, is employed to formulate the ranking of mAb aggregation. ThT binding can determine the propensity of proteins to aggregate in a few days, illustrating that ThT binding would be a valuable screening tool.
Assuntos
Anticorpos Monoclonais/química , Cromatografia em Gel/métodos , Proteínas/química , Tiazóis/química , Benzotiazóis , Química Farmacêutica/métodos , Corantes Fluorescentes/química , Dobramento de Proteína , Estabilidade ProteicaRESUMO
Understanding antibody aggregation is of great significance for the pharmaceutical industry. We studied the aggregation of five different therapeutic monoclonal antibodies (mAbs) with size-exclusion chromatography-high-performance liquid chromatography (SEC-HPLC), fluorescence spectroscopy, electron microscopy, and light scattering methods at various temperatures with the aim of gaining insight into the aggregation process and developing models of it. In particular, we find that the kinetics can be described by a second-order model and are non-Arrhenius. Thus, we develop a non-Arrhenius model to connect accelerated aggregation experiments at high temperature to long-term storage experiments at low temperature. We evaluate our model by predicting mAb aggregation and comparing it with long-term behavior. Our results suggest that the number of monomers and mAb conformations within aggregates vary with the size and age of the aggregates, and that only certain sizes of aggregates are populated in the solution. We also propose a kinetic model based on conformational changes of proteins and monomer peak loss kinetics from SEC-HPLC. This model could be employed for a detail analysis of mAb aggregation kinetics.
Assuntos
Anticorpos Monoclonais/química , Modelos Químicos , Tecnologia Farmacêutica/métodos , Anticorpos Monoclonais/uso terapêutico , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Guanidina/química , Cinética , Luz , Microscopia Eletrônica de Transmissão , Conformação Proteica , Dobramento de Proteína , Multimerização Proteica , Espalhamento de Radiação , Espectrometria de Fluorescência , Espectrofotometria Ultravioleta , TemperaturaRESUMO
Monoclonal antibodies are the fastest growing class of biologics in the pharmaceutical industry. The correlation between mAb glycosylation and aggregation has not been elucidated in detail, yet understanding the structure-stability relationship involving glycosylation is critical for developing successful drug formulations. We conducted studies of temperature-induced aggregation and compared the stability of both glycosylated and aglycosylated forms of a human IgG1. In parallel, we also performed molecular dynamics simulations of the glycosylated full antibody to gain an understanding of the polysaccharide surroundings at the molecular level. Aglycosylated mAbs are somewhat less stable and therefore aggregate more easily than the glycosylated form at the temperatures studied. Glycosylation seems to enhance solubility and stability of these therapeutics and thus might be important for long-term storage.
Assuntos
Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/química , Glicosilação , Humanos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Estabilidade Proteica , TemperaturaRESUMO
Since each antibody has its unique physical chemical properties, optimal formulation for one antibody is likely not applicable for the others. To rapidly screen multiple antibody formulations, an automated system was constructed to perform sample preparation, testing, and data management. Using the automatic system, up to 500 liquid formulations can be prepared in deep well microplates and further distributed into standard microplates that can be stored under different stress conditions for degradation studies. In addition, the system can also be used to prepare samples in microplates for different analytical measurements such as UV spectroscopy, turbidity, dynamic light scattering (DLS), SEC-HPLC, RP-HPLC and CEX-HPLC, and automated lab-on-a-chip platform (ALP). The data generated using different techniques in the automatic system were comparable to those of the classical approaches.
Assuntos
Anticorpos Monoclonais/análise , Composição de Medicamentos/métodos , Ensaios de Triagem em Larga Escala/métodos , Laboratórios , Preparações Farmacêuticas/análise , Robótica , Composição de Medicamentos/instrumentação , Estabilidade de Medicamentos , Ensaios de Triagem em Larga Escala/instrumentação , Estabilidade ProteicaRESUMO
Protein-carbohydrate interactions are important for glycoprotein structure and function. Antibodies of the IgG class, with increasing significance as therapeutics, are glycosylated at a conserved site in the constant Fc region. We hypothesized that disruption of protein-carbohydrate interactions in the glycosylated domain of antibodies leads to the exposure of aggregation-prone motifs. Aggregation is one of the main problems in protein-based therapeutics because of immunogenicity concerns and decreased efficacy. To explore the significance of intramolecular interactions between aromatic amino acids and carbohydrates in the IgG glycosylated domain, we utilized computer simulations, fluorescence analysis, and site-directed mutagenesis. We find that the surface exposure of one aromatic amino acid increases due to dynamic fluctuations. Moreover, protein-carbohydrate interactions decrease upon stress, while protein-protein and carbohydrate-carbohydrate interactions increase. Substitution of the carbohydrate-interacting aromatic amino acids with non-aromatic residues leads to a significantly lower stability than wild type, and to compromised binding to Fc receptors. Our results support a mechanism for antibody aggregation via decreased protein-carbohydrate interactions, leading to the exposure of aggregation-prone regions, and to aggregation.
Assuntos
Metabolismo dos Carboidratos , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Aminoácidos/metabolismo , Sequência de Carboidratos , Carboidratos/química , Linhagem Celular , Simulação por Computador , Resposta ao Choque Térmico , Humanos , Interações Hidrofóbicas e Hidrofílicas , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Peptídeos/metabolismo , Ligação Proteica , Estabilidade Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Propriedades de SuperfícieRESUMO
The large size and complexity of many proteins constrains the reversed-phase high-performance liquid chromatography packings that are useful for their separation. Wide-pore, superficially porous, silica-based packings with solid 4.5-microm cores and a 0.25-microm porous outer layer (Poroshell) demonstrate a variety of characteristics that are beneficial for the separation of proteins. A shorter diffusion distance allows separations of large molecules at high linear velocities. This benefit over totally porous particles is clearly shown using separations of a peptide-protein standard. The structure and reduced surface area (4.5 m2/g) of these superficially porous particles simplifies interactions with its surface, resulting in improved peak shapes and resolution. Specialized bonding chemistries for low- and high-pH operation may be used to change band-spacing and achieve atypical separations. These rapid analysis options are demonstrated using protein standards and very high molecular weight glycosylated proteins including intact monoclonal antibodies, IgM, alpha2-macroglobulin, and glycophorin. In liquid chromatography-mass spectrometry analysis of a myoglobin peptide digest, bidentate-C18-bonded superficially porous packings achieve complete runs in 4 min and demonstrate an elution pattern that is unique from that of material bonded with sterically protected C18 ligands.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Peptídeos/análise , Proteínas/análise , Anticorpos Monoclonais/análise , Cromatografia Líquida de Alta Pressão/instrumentação , Glicoforinas/análise , Imunoglobulina M/análise , Espectrometria de Massas/instrumentação , Espectrometria de Massas/métodos , Tamanho da Partícula , Porosidade , Reprodutibilidade dos Testes , alfa-Macroglobulinas/análiseRESUMO
Previous studies on the effect of glycosylation on the elimination rate of antibodies have produced conflicting results. Here, we performed pharmacokinetic studies in mice with two preparations of a monoclonal IgG1 antibody enriched for complex type or high mannose type oligosaccharides at the Fc glycosylation site. No significant difference in the serum half-life was found between the two antibody glycoforms, nor was any difference observed in the serum half-lives of different complex type glycoforms. To evaluate the influence of glycosylation within the variable domain, a second monoclonal antibody, glycosylated in both the Fc and Fv domains, was separated into fractions containing different amounts of Fv-associated sialic acid and administered to mice. Again, no significant difference was found in the clearance rates of variants carrying different amounts of Fv-associated sialic acid or lacking Fv-glycosylation. These results suggest that glycosylation has little or no impact on the pharmacokinetic behavior of these two monoclonal antibodies in mice.
Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Animais , Anticorpos Monoclonais/farmacologia , Glicosilação , Humanos , Imunoglobulina G/farmacologia , CamundongosRESUMO
The inter-heavy-chain disulfide bonds of the IgG4 subclass can be described as being at equilibrium with the intra-chain disulfide bonds. This means that a fraction of IgG4 has noncovalently linked heavy chains (half-antibody). The percentage of half-antibodies produced depends upon the expression system used. Nondenaturing assays fail to separate the half-antibodies from the native form because two half-molecules are held together by noncovalent forces. The pharmaceutical industry needs a reliable denaturing assay for checking batch-to-batch consistency. Until now sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) has been the standard method used to detect the presence of half-antibodies. However, this technique is laborious and cannot be automated. Furthermore, cumbersome densitometric measurements are necessary for quantification. To overcome these disadvantages a chip-based gel electrophoresis method was investigated. In the nonreduced format the separation profile is compared with that from SDS-PAGE. The limit of quantification as a percentage of the amount applied, repeatability, reproducibility, and linearity are compared with those of SDS-PAGE. The amounts of half-antibody and of by- and degradation products were determined for several batches by using area percentage and by external calibration with IgG4 as a reference standard. Both methods allow avoidance of error introduction for the quantification as is the case by application of myosin as reference concentration. Both sets of results are compared with each other and with the results from SDS-PAGE. In the reduced format it is noted that the reduction of the inter-heavy-chain disulfide bridges proceeds faster than the reduction of the heavy-light-chain bonds. Therefore optimized conditions are necessary to obtain a complete reduction.