Abstract
The project aims to tackle recombinant protein Quality Control by means of Analytical Quality by Design (AQbD). Recombinant proteins are complex molecules displaying several conformations, post-translational modifications and microheterogeneity which affect efficacy and safety when used as biopharmaceuticals. Liquid chromatography, capillary electrophoresis and mass spectrometry will be applied for evaluation of Critical Quality Attributes (e.g., charge and mass variants) of some monoclonal antibodies (mAbs). Infliximab is selected to establish appropriate validation protocols; in this regard the AQbD is applied providing a rational approach that combines the optimization and validation steps and allows the identification of an optimal zone (the design space) where all possible combinations of method parameters lead to optimal performance. An analytical platform based on the achieved results will be created and applied to a variety of mAbs. 1. Project Framework and Objectives The primary objective of the project was to translate modern regulatory updates, specifically the risk management and development principles outlined in the ICH Q14 and ICH Q9(R1) guidelines, into practical, robust laboratory operations. By adopting an Analytical Quality-by-Design (AQbD) approach combined with Design of Experiments (DoE), the consortium aimed to develop validated, reliable, and versatile Platform Analytical Procedures (PAPs). These platforms target the advanced characterization of charge and mass variants of therapeutic recombinant proteins and monoclonal antibodies (mAbs), which serve as critical quality attributes (CQAs) for biosimilarity evaluation and industrial quality control. The workflow was successfully implemented through the coordinated efforts of three operational units: RU1: University of Florence (Principal Investigator Unit). RU2: Alma Mater Studiorum – University of Bologna. RU3: University of Pavia. Infliximab was selected as the model mAb due to its extensive documentation in literature, providing an ideal benchmark for method validation. 2. Core Technical Achievements by Operational Units RU1 spearheaded the regulatory-compliant framework, defining the overarching AQbD roadmap and supervising the multivariate statistical processing across the project. Focusing on imaged capillary IsoElectric Focusing (icIEF) carried out at the Istituto Superiore di Sanità - ISS, the unit defined an Analytical Target Profile (ATP) requiring a robust PAP capable of measuring the apparent isoelectric point (pI) of mAb isoforms with a bias of less than 4%. The separation conditions were optimized by developing a cause-effect matrix hazard and succesively by DoE; the concentration levels of the components of the background electrolyte i.e., iminodiacetic acid and L-Arginine as well as the specific mixture composition of carrier ampholytes (CAs) were found to exert the predominant effects on the Critical Procedure Attributes (CPAs). Through Response Surface Methodology and Monte Carlo simulations, a Method Operable Design Region (MODR) was defined using as the optimal blend a mixture of 2% CA pH 3–10, 2% CA pH 5–8, and 2% CA pH 8–10.5. Under these conditions, the marker measurement bias was below the 2% threshold. Our unit at UniBo successfully transitioned the AQbD workflow into conventional capillary isoelectric focusing (cIEF). Through initial brainstorming and a cause-effect matrix, the multiple potential Critical Procedure Parameters (pCPPs), including methyl cellulose, urea, stabilizers, and capillary coatings were considered. Using a mixture design, the CAs interactions were evaluated and a robust MODR was defined. The optimized condition was established using a specific CA blend of 1.5% at pH 3–10, 1.5% at pH 5–8, and 2.0% at pH 8–10.5. Method validation yielded exceptional robustness, showing a pI measurement bias below 0.3%. Furthermore, a cross-platform comparison with the icIEF data from the ISS demonstrated total alignment, proving the excellent accuracy, linearity, trueness, and precision of our conventional method. Finally, we verified the horizontal applicability of this robust platform by successfully profiling the distinct charge variants of other therapeutic proteins, namely Bevacizumab and Daratumumab. RU3 successfully focused on establishing a validated Size Exclusion Chromatography with UV detection (SEC-UV) for evaluating mass heterogeneity (high molecular weight aggregates and low molecular weight fragments) under the AQbD paradigm, aiming to secure bioproduct purity and stability tracking. Utilizing DoE, the unit systematically evaluated mobile phase variations, such as salt concentration, pH, organic modifiers, and amino acid additives, to control secondary hydrophobic and electrostatic interactions between the analyte and the stationary phase. The study proved that column selection is the most critical parameter in achieving a robust, validated MODR for co-eluting peak pairs (monomer and low molecular weight fragments). 3. Strategic Collaborations and Industrial Attraction A major outcome of the project was its capacity to attract substantial external industrial interest and private funding, ensuring high-level technology transfer: Agilent Technologies Agreement (RU2): A formal Research Collaboration Agreement signed in April 2025 provided our laboratory at UniBo with a state-of-the-art Capillary Electrophoresis instrument valued at €104,000 free of charge, which was vital for consolidating our cIEF platform validation. This instrument has now been permanently purchased by our Department (FaBiT UniBo) under preferential conditions, securing the long-term continuity of this high-level analytical line. Waters Agreement (RU3): A collaboration agreement brought a state-of-the-art bio-inert Alliance iS Bio HPLC System to RU3, drastically reducing unwanted secondary interactions, minimizing peak tailing, and accelerating reproducible size variant analysis. Brogioni Foundation Grant (RU1): External funding of €40,000 was secured to launch a project (named SoFar), which utilizes the developed analytical expertise to track the stability of mAbs. 4. Educational, Socio-Generational, and Dissemination The consortium strictly supported equal opportunities, integrating early-stage female researchers into core positions. This included a female research fellow at UniBo (RU2) whose high-level training was extended for 5 additional months using internal departmental funds to ensure project sustainability. Awards: The high industrial relevance of the project was highlighted when the research team member, Virginia Ghizzani, was honored with the first edition of the prestigious "Alessandro Rigamonti Prize" (€5,000) by the Associazione Farmaceutici Industria (AFI). Results have been widely disseminated via peer-reviewed papers, alongside numerous oral and keynote presentations (a total of 12) and poster communications (a total of 6) at major international symposia (e.g., Geneva, Shanghai, Bruges, and Riva del Garda). List of the the published papers 1) V. Ghizzani, S. Orlandini, A. Ascione, B. Pasquini, S. Tengattini, C. Temporini, R. Gotti, G. Massolini, S. Furlanetto, F. Luciani, “A Design of Experiments and Risk Management Driven Analytical Platform for Charge Variant Analysis of Therapeutic Antibodies by Imaged Capillary Isoelectric Focusing", AAPS J. 28 (2026) 27. https://doi.org/10.1208/s12248-025-01178-8 2) V. Ghizzani, A. Ascione, F. Gonnella, G. Massolini, F. Luciani, “Exploring imaged capillary isoelectric focusing parameters for enhanced charge variants quality control.” Front. Chem. 13, 1536222. (2025). https://doi.org/10.3389/fchem.2025.1536222 3) L. Floris, B. Pasquini, S. Orlandini, F. Luciani, G. Massolini, S. Furlanetto, R. Gotti, “Systematic development and optimization of a cIEF method for the charge variant analysis of moderately basic monoclonal antibodies using the Analytical Quality by Design strategy”. J. Pharm. Biomed. Anal. 279 (2026) 117576. https://doi.org/10.1016/j.jpba.2026.117576 5. Sustainability and Predictive Analysis Crucially, our validated cIEF-AQbD platform is being deployed within the SoFar project to run real-world chemical and physical stability tests on opened vials and prepared syringes of high-cost oncology drugs, such as Daratumumab. Extending multi-day stability data will eliminate the costly disposal of residual doses in hospital settings. This direct pharmacoeconomic translation allows public healthcare systems to minimize waste and reinvest vital financial resources into enhancing patient care quality.
Project details
Unibo Team Leader: Roberto Gotti
Unibo involved Department/s:
Dipartimento di Farmacia e Biotecnologie
Coordinator:
Università degli Studi di FIRENZE(Italy)
Total Unibo Contribution: Euro (EUR) 59.976,00
Project Duration in months: 24
Start Date:
28/09/2023
End Date:
28/02/2026
