Biopharmaceuticals, monoclonal antibody (mAb)-based therapeutics in particular, have positively impacted millions of lives. MAbs and related therapeutics are highly desirable from a biopharmaceutical perspective as they are highly target specific and well tolerated within the human system. Nevertheless, several mAbs have been discontinued or withdrawn based either on their inability to demonstrate efficacy and/or due to adverse effects. Approved monoclonal antibodies and derived therapeutics have been associated with adverse effects such as immunogenicity, cytokine release syndrome, progressive multifocal leukoencephalopathy, intravascular haemolysis, cardiac arrhythmias, abnormal liver function, gastrointestinal perforation, bronchospasm, intraocular inflammation, urticaria, nephritis, neuropathy, birth defects, fever and cough to name a few. The advances made in this field are also impeded by a lack of progress in bioprocess development strategies as well as increasing costs owing to attrition, wherein the lack of efficacy and safety accounts for nearly 60 % of all factors contributing to attrition. This reiterates the need for smarter preclinical development using quality by design-based approaches encompassing carefully designed predictive models during early stages of drug development. Different in vitro and in silico methods are extensively used for predicting biological activity as well as toxicity during small molecule drug development; however, their full potential has not been utilized for biological drug development. The scope of in vitro and in silico tools in early developmental stages of monoclonal antibody-based therapeutics production and how it contributes to lower attrition rates leading to faster development of potential drug candidates has been evaluated. The applicability of computational toxicology approaches in this context as well as the pitfalls and promises of extending such techniques to biopharmaceutical development has been highlighted.

Monoclonal antibodies (mAbs) constitute a rapidly growing biopharmaceutical sector. However, their growth is impeded by high failure rates originating from failed clinical trials and developability issues in process development. There is, therefore, a growing need for better in silico tools to aid in risk assessment of mAb candidates to promote early-stage screening of potentially problematic mAb candidates. In this study, a quantitative structure–activity relationship (QSAR) modelling workflow was designed for the prediction of hydrophobic interaction chromatography (HIC) retention times of mAbs. Three novel descriptor sets derived from primary sequence, homology modelling, and atomistic molecular dynamics (MD) simulations were developed and assessed to determine the necessary level of structural resolution needed to accurately capture the relationship between mAb structures and HIC retention times. The results showed that descriptors derived from 3D structures obtained after MD simulations were the most suitable for HIC retention time prediction with a R² = 0.63 in an external test set. It was found that when using homology modelling, the resulting 3D structures became biased towards the used structural template. Performing an MD simulation therefore proved to be a necessary post-processing step for the mAb structures in order to relax the structures and allow them to attain a more natural conformation. Based on the results, the proposed workflow in this paper could therefore potentially contribute to aid in risk assessment of mAb candidates in early development.

Introduction: Monoclonal antibodies (mAbs) are important therapeutics. However, the enhanced potential for aggregation has become a critical quality parameter during the production of mAbs. Furthermore, mAb aggregation may also present a potential health risk in a clinical setting during the administration of mAb therapeutics to patients. While the extent of immunotoxicity in patient populations is uncertain, reports show it can lead to immune responses via cell activation and cytokine release. In this study, an autologous in vitro skin test designed to predict adverse immune events, including skin sensitization, was used as a novel assay for the assessment of immunotoxicity caused by mAb aggregation. Material and Methods: Aggregation of mAbs was induced by a heat stress protocol, followed by characterization of protein content by analytical ultra-centrifugation and transmission electron microscopy, revealing a 4% aggregation level of total protein content. Immunotoxicity and potential skin sensitization caused by the aggregates, were then tested in a skin explant assay. Results: Aggregated Herceptin and Rituximab caused skin sensitization, as shown by histopathological damage (grade II–III positive response) together with positive staining for Heat Shock Protein 70 (HSP70). Changes in T cell proliferation were not observed. Cytokine analysis revealed a significant increase of IL-10 for the most extreme condition of aggregation (65 °C at pH3) and a trend for an overall increase of IFN-γ, especially in response to Rituximab. Conclusions: The skin explant assay demonstrated that aggregated mAbs showed adverse immune reactions, as demonstrated as skin sensitization, with histopathological grades II-III. The assay may, therefore, be a novel tool for assessing immunotoxicity and skin sensitization caused by mAb aggregation.

Monoclonal antibody (mAb) therapeutics have a promising outlook within the pharmaceutical industry having made positive strides in both research and development as well as commercialisation, however this development has been hampered by manufacturing failures and attrition. This study explores the applicability of traditional in vitro toxicity tests for detecting any off-target adverse effect elicited by mAbs on specific organ systems using hepatocarcinoma cell line (HepG2) and human dermal fibroblasts neonatal (HDFn), respectively. The mechanism of antibody dependent cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) via complement activation, and complement dependent cellular cytotoxicity (CDCC) were assessed. Major results: no apparent ADCC, CDCC, or CDC mediated decrease in cell viability was measured for HepG2 cells. For HDFn cells, though ADCC or CDCC mediated decreases in cell viability wasn’t detected, a CDC mediated decrease in cell viability was observed. Several considerations have been elucidated for development of in vitro assays better suited to detect off target toxicity of mAbs.