The critical quality attributes of a given biotherapeutic monoclonal antibody (mAb), its molecular characterization, functional assessment and effector function analysis should be defined and profiled in detail during the life cycle of a biotherapeutic drug. In the past, this product characterization was simple and standardized. In today’s complex world of biologics, success demands a more thoughtful approach and drug developers are investing in advanced analytics much earlier in the development process. Indeed, investing in selected sophisticated and state of the art analytics in early developmental phases of a therapeutic monoclonal antibody may mitigate risks by confirming that the drug candidate has the required basic characteristics and functionality. Therefore, recognizing the added value of an early functional characterization of new biological entities (NBE) in the pharmaceutical industry, this work was focused on the development and qualification of novel methodologies defining the proper analytical characterization panel for a new therapeutic monoclonal antibody. In details, this PhD project was specifically designed to identify, develop and qualify with a stepwise approach in silico tools and in vitro assays aimed at studying the biological activity, predictive pharmacokinetics (PK) and potential immunogenicity of therapeutic drugs in early phase. To this aim Anti-TIGIT mAb was selected as proof of concept molecule. First, an investigation on the mechanism of action of Anti-TIGIT has been carried out and then different approaches were applied to identify the best in vitro assay reflecting the mechanism underlying Anti-TIGIT biological effect. Once selected as the best method a cell-based Anti-TIGIT ligand binding bioassay, it was checked for linearity, precision, accuracy and specificity demonstrating its fitting for the purpose. Different alternative assays for the assessment of the mAb’s effector functions were also presented and qualified showing the ability of Anti-TIGIT to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Then, the attention was focused on the predictive pharmacokinetics of Anti-TIGIT and specifically on in vitro techniques to assess affinity and kinetic of the interaction with neonatal Fc receptor (FcRn) demonstrating that the developed and qualified methods were reliable, precise and accurate. Moreover, forced degradation studies, where the mAb underwent different stresses, were performed demonstrating the assays’ suitability for the purpose highlighting the impact of the applied stresses on both Anti-TIGIT biological activity and PK. In addition, to ensure a good comparison and integration of all the findings, several orthogonal assays were also developed, and the results compared demonstrating the equivalence of the methods. In the end, also an in silico tool was used to predict the potential immunogenicity of the drug. Altogether the findings of this work elucidate and integrate the strategies applied in different pharmaceutical companies for method development and validation and extend the knowledge on the methodologies used for therapeutic monoclonal antibodies characterization and stability studies opening new perspectives for the industrial setting.

(2021). DEVELOPMENT & QUALIFICATION OF BIOASSAYS FOR THE DETERMINATION OF THE BIOACTIVITY, PREDICTIVE PHARMACOKINETICS AND POTENTIAL IMMUNOGENICITY OF THERAPEUTIC ANTIBODIES.

DEVELOPMENT & QUALIFICATION OF BIOASSAYS FOR THE DETERMINATION OF THE BIOACTIVITY, PREDICTIVE PHARMACOKINETICS AND POTENTIAL IMMUNOGENICITY OF THERAPEUTIC ANTIBODIES

ANDERLONI, Giulia
2021-05-05

Abstract

The critical quality attributes of a given biotherapeutic monoclonal antibody (mAb), its molecular characterization, functional assessment and effector function analysis should be defined and profiled in detail during the life cycle of a biotherapeutic drug. In the past, this product characterization was simple and standardized. In today’s complex world of biologics, success demands a more thoughtful approach and drug developers are investing in advanced analytics much earlier in the development process. Indeed, investing in selected sophisticated and state of the art analytics in early developmental phases of a therapeutic monoclonal antibody may mitigate risks by confirming that the drug candidate has the required basic characteristics and functionality. Therefore, recognizing the added value of an early functional characterization of new biological entities (NBE) in the pharmaceutical industry, this work was focused on the development and qualification of novel methodologies defining the proper analytical characterization panel for a new therapeutic monoclonal antibody. In details, this PhD project was specifically designed to identify, develop and qualify with a stepwise approach in silico tools and in vitro assays aimed at studying the biological activity, predictive pharmacokinetics (PK) and potential immunogenicity of therapeutic drugs in early phase. To this aim Anti-TIGIT mAb was selected as proof of concept molecule. First, an investigation on the mechanism of action of Anti-TIGIT has been carried out and then different approaches were applied to identify the best in vitro assay reflecting the mechanism underlying Anti-TIGIT biological effect. Once selected as the best method a cell-based Anti-TIGIT ligand binding bioassay, it was checked for linearity, precision, accuracy and specificity demonstrating its fitting for the purpose. Different alternative assays for the assessment of the mAb’s effector functions were also presented and qualified showing the ability of Anti-TIGIT to induce antibody-dependent cell-mediated cytotoxicity (ADCC). Then, the attention was focused on the predictive pharmacokinetics of Anti-TIGIT and specifically on in vitro techniques to assess affinity and kinetic of the interaction with neonatal Fc receptor (FcRn) demonstrating that the developed and qualified methods were reliable, precise and accurate. Moreover, forced degradation studies, where the mAb underwent different stresses, were performed demonstrating the assays’ suitability for the purpose highlighting the impact of the applied stresses on both Anti-TIGIT biological activity and PK. In addition, to ensure a good comparison and integration of all the findings, several orthogonal assays were also developed, and the results compared demonstrating the equivalence of the methods. In the end, also an in silico tool was used to predict the potential immunogenicity of the drug. Altogether the findings of this work elucidate and integrate the strategies applied in different pharmaceutical companies for method development and validation and extend the knowledge on the methodologies used for therapeutic monoclonal antibodies characterization and stability studies opening new perspectives for the industrial setting.
biotherapeutic antibodies; bioassays; method development and qualification; biological activity; predictive pharmacokinetics; potential immunogenicity.
(2021). DEVELOPMENT & QUALIFICATION OF BIOASSAYS FOR THE DETERMINATION OF THE BIOACTIVITY, PREDICTIVE PHARMACOKINETICS AND POTENTIAL IMMUNOGENICITY OF THERAPEUTIC ANTIBODIES.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/10447/498154
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