RESUMO
Strongly attractive self-interaction of therapeutic protein candidates can impose challenges for manufacturing, filling, stability, and administration due to elevated viscosity or aggregation propensity. Suitable formulations can mitigate these issues to a certain extent. Understanding the self-interaction mechanism on a molecular basis and rational protein engineering provides a more fundamental approach, and it can save costs and efforts as well as alleviate risks at later stages of development. In this study, we used computational methods for the identification of aggregation-prone regions in a mAb and generated mutants based on these findings. We applied hydrogen-deuterium exchange mass spectrometry to identify distinct self-interaction hot spots. Ultimately, we generated mAb variants based on a combination of both approaches and identified mutants with low attractive self-interaction propensity, minimal off-target binding, and even improved target binding. Our data show that the introduction of arginine in spatial proximity to hydrophobic patches is highly beneficial on all these levels. For our mAb, variants that contain more than one aspartate residue flanking to the hydrophobic HCDR3 show decreased attractive self-interaction at unaffected off-target and target binding. The combined engineering strategy described here underlines the high potential of understanding self-interaction in the early stages of development to predict and reduce the risk of failure in subsequent development.
Assuntos
Anticorpos Monoclonais/genética , Mutação/genética , Linhagem Celular Tumoral , Medição da Troca de Deutério/métodos , Humanos , Interações Hidrofóbicas e Hidrofílicas , Espectrometria de Massas/métodos , Engenharia de Proteínas/métodos , ViscosidadeRESUMO
PURPOSE: To speed up the drug development process in the biopharmaceutical industry, high throughput methods are indispensable for assessing drug candidates and potential lead formulations, in particular during late stages of discovery and early phases of development. This study aimed to establish a bio-layer-interferometry based high throughput assay for assessing formulation dependent mAb self-interaction (SI-BLI) and to compare the results with kD values obtained by dynamic light scattering (DLS). METHODS: Self-interaction of proprietary and commercially available mAbs was analyzed by SI-BLI and dynamic light scattering (DLS). RESULTS: We found significant correlations of the SI-BLI results and kD-values obtained by DLS for both, different mAbs in one platform formulation and for mAbs formulated in several buffer compositions. In total, we assessed self-interaction propensity of different mAbs in 58 formulations and found significant Pearson correlation (p < 0.05) between kD and results of SI-BLI. CONCLUSIONS: The SI-BLI results correlate with kD and enable fast ranking of both different drug candidates and potential lead formulations. Thus, SI-BLI might decrease the risk to lose potent mAb candidates during transition from discovery to development, and help to accelerate the development of high concentration liquid formulations.
Assuntos
Adalimumab/química , Omalizumab/química , Composição de Medicamentos , Ensaios de Triagem em Larga Escala , Humanos , Interferometria/métodos , Cinética , Ligação Proteica , Conformação Proteica , Multimerização ProteicaRESUMO
Histone deacetylase (HDAC) inhibitors have been exploited for years to improve recombinant protein expression in mammalian production cells. However, global HDAC inhibition is associated with negative effects on various cellular processes. microRNAs (miRNAs) have been shown to regulate gene expression in almost all eukaryotic cell types by controlling entire cellular pathways. Since miRNAs recently have gained much attention as next-generation cell engineering tool to improve Chinese hamster ovary (CHO) cell factories, we were interested if miRNAs are able to specifically repress HDAC expression in CHO cells to circumvent limitations of unspecific HDAC inhibition. We discovered a novel miRNA in CHO cells, miR-2861, which was shown to enhance productivity in various recombinant CHO cell lines. Furthermore, we demonstrate that miR-2861 might post-transcriptionally regulate HDAC5 in CHO cells. Intriguingly, siRNA-mediated HDAC5 suppression could be demonstrated to phenocopy pro-productive effects of miR-2861 in CHO cells. This supports the notion that miRNA-induced inhibition of HDAC5 may contribute to productivity enhancing effects of miR-2861. Furthermore, since product quality is fundamental to safety and functionality of biologics, we examined the effect of HDAC inhibition on critical product quality attributes. In contrast to unspecific HDAC inhibition using VPA, enforced expression of miR-2861 did not negatively influence antibody aggregation or N-glycosylation. Our findings highlight the superiority of miRNA-mediated inhibition of specific HDACs and present miR-2861 as novel cell engineering tool for improving CHO manufacturing cells.
Assuntos
Regulação da Expressão Gênica , Inibidores de Histona Desacetilases/metabolismo , Engenharia Metabólica , MicroRNAs/metabolismo , Proteínas Recombinantes/biossíntese , Animais , Anticorpos/genética , Anticorpos/metabolismo , Células CHO , Cricetulus , Proteínas Recombinantes/genéticaRESUMO
Attractive self-interaction processes in antibody formulations increase the risk of aggregation and extraordinarily elevated viscosity at high protein concentrations. These challenges affect manufacturing and application. This study aimed to understand the self-interaction process of Infliximab as a model system with pronounced attractive self-interaction. The association mechanism was studied by a multi-method approach comprising analytical ultracentrifugation, dynamic light scattering, small angle X-ray scattering, self-interaction bio-layer interferometry and hydrogen-deuterium exchange mass spectrometry. Based on our results, both Fab and Fc regions of Infliximab are involved in self-interaction. We hypothesize a mechanism based on electrostatic interactions of polar and charged residues within the identified areas of the heavy and the light chain of the mAb. The combination of fast and reliable screening methods and low throughput but high resolution methods can contribute to detailed characterization and deeper understanding of specific self-interaction processes.