Investigation of lyophilized antibody formulations to enable short freeze-drying cycles and storage at room temperature

Investigation of lyophilized antibody formulations to enable short freeze-drying cycles and storage at room temperature: Antibodies are dominating the biopharmaceutical market and are expected to grow further. Aiming to improve existing treatments, new antibody derivatives of improved efficacy and safety are being developed in a competitive market. Antibody derivatives include but are not limited to antibody-drug conjugates and Fc-fusion proteins. Due to their higher complexity, they are often less stable as liquids, increasing the demand for lyophilized formulations to ensure protein storage stability over the desired shelf life. In many cases, these antibody formats require lower doses, posing challenges to formulation and freeze-drying process development. Commercialized lyophilized antibodies typically contain disaccharides, most frequently sucrose, as a stabilizer and bulking agent. The low glass transition temperature of sucrose requires time- and energy-intensive, thus expensive freeze-drying cycles. At lower protein concentrations, this becomes even more relevant, raising the risk of product collapse during freeze-drying. Collapse occurs when primary drying is performed above the glass transition temperature or collapse temperature of the formulation. It is current dogma to design freeze-drying cycles that provide pharmaceutically elegant lyophilisates as collapse leads to batch inhomogeneity causing rejects, higher complaint rates, and most importantly may potentially be detrimental to protein storage stability. Thus, there is a need to look into alternative excipients for future freeze-dried antibody formulations. The presented work investigated amorphous excipients to be used as alternative excipients to sucrose for freeze-dried antibody formulations, increasing the formulation’s glass transition temperature. The main objectives were to investigate their ability to render pharmaceutically elegant lyophilisates upon short freeze-drying cycles, and to stabilize antibodies during freeze-drying and subsequent storage. Special focus was given to storage stability at elevated temperatures with the aim to study the potential for room temperature stable formulations. At first, an imaging technique was established to evaluate the impact of excipients and freeze-drying cycles on cake appearance and structure. Different imaging techniques were compared regarding qualitative and quantitative characterization of the entire lyophilisate, and their potential for non-invasive evaluation of structure and morphology in the glass vial (Chapter 1). The comparative analysis revealed limitations of scanning electron microscopy, the current state of the art technique to characterize cake morphology. Micro-computed tomography was introduced as a technique allowing for comprehensive and reproducible imaging of cake structure and morphology. Having established a method for evaluation of cake appearance, the next step of this work was focused on formulation development. Dextrans of different molecular weight from 1 to 500 kDa (Chapter 2) followed by HPBCD-based formulations in combination with other amorphous compounds (Chapter 3) were investigated. Their impact on thermal properties, cake appearance, other physico-chemical product quality attributes, and protein stability of two model antibodies was characterized. In particular, HPBCD was found to be a promising excipient, while dextran showed several limitations. Large dextrans of 40 kDa or higher were shown to increase the viscosity of the formulations leading to long reconstitution times, and did not sufficiently stabilize the antibodies during freeze-drying compared to smaller dextrans and HPBCD. The work highlighted limitations of dextrans with regards to protein stability, due to antibody glycation during storage at elevated temperatures. HPBCD rendered lyophilisates with good product quality attributes and ensured antibody stability during freeze-drying and even at elevated storage temperatures. Best antibody stability was obtained in combination with sucrose, highlighting the fact that disaccharides will remain a mandatory part of freeze-dried antibody formulations. To further maximize protein stability, a thorough characterization of the optimal ratio of HPBCD and sucrose will be essential. These formulations which provided good stability and product quality attributes were subsequently used for freeze-drying process optimization (Chapter 4). Primary drying parameters were optimized for a short freeze-drying cycle that renders pharmaceutically elegant lyophilisates. The presented work demonstrated that amorphous excipients with higher glass transition temperatures allow for shorter freeze-drying cycles while providing lyophilisates with improved cake appearance. Ultimately, the HPBCD-based formulation with addition of sucrose enabled the development of a short, single-step freeze-drying cycle while maintaining pharmaceutically elegant lyophilisates eventually reducing cycle time by 50%. Overall, the current work demonstrated the potential of alternative amorphous excipients, which in contrast to crystalline bulking agents contribute to protein stability while avoiding additional complexity in the freeze-drying cycle. The combined use of HPBCD with sucrose may provide a formulation for low concentrated protein formulations that enables the development of short freeze-drying cycles while maintaining pharmaceutically elegant lyophilisates. The presented work may encourage considerations to store freeze-dried formulations at (controlled) room temperature rather than refrigerated conditions in the future.