Downstream process development and preliminary formulation development for the bispecific antibody NF-CU N297Q for a clinical phase I/IIa trial

Abstract

Over the last two decades, monoclonal antibodies improved the treatment options for various cancers significantly. However, the production process for these proteinbased therapeutics is considerably more demanding than that for small molecule drugs. This is particularly true for complex proteins such as bispecific antibodies. The purpose of this thesis was to develop a production process and a preliminary formulation buffer for the bispecific FLT3ˆCD3 antibody NF-CUN297Q, which was developed for the treatment of acute myeloid leukemia (AML) and is intended to be applied in a clinical phase I/IIa trial. Thus, the main challenges for the production- and formulation development are to produce a sufficient amount of the active pharmaceutical ingredient (API) with very high quality and to ensure long-term stability of the product. Good manufacturing practice (GMP)-compliant production processes are usually developed and performed by the pharmaceutical industry. The special aspect of this thesis is the realization of GMP-compliant late-stage development of a protein-based pharmaceutical drug at a small spin-off company operating at an university campus. As a first step, specifications for critical quality attributes (CQA) were established and justified. If mandatory requirements from regulatory authorities existed for certain CQAs, these specifications were adopted for the development process. If no specific requirements were demanded, commonly applied specifications were adopted from similar production processes. If commonly applied specifications were not feasible, specifications were deducted from experimental characterizations. One example for this approach is the determination of the tolerable amount of product-related impurities such as aggregates, light chain dimers and light chains in the final product formulation. The required amount of the API for a clinical phase I/IIa trial including all necessary validations was discussed and determined theoretically. The second task was to find an economic approach for the development of a production process which fulfills the previously developed specifications and complies with GMP requirements. At the beginning of process development, a suitable Chinese hamster ovary (CHO)-based expression cell line, a so-called primary seed bank (PSB), was generated and a scalable standard fermentation protocol was successfully adopted. A variety of purification applications was tested for their suitability to fulfill the previously determined specifications. In conclusion, a production process consisting of affinity chromatography using KappaSelect followed by anion exchangeand hydrophobic interaction chromatography and nanofiltration was developed. The small-scale model (scale 1:100) of the process fulfills the requirements for product quality and quantity and complies with GMP requirements. A preliminary protein formulation buffer for the purified bispecific antibody was selected using thermodynamic and kinetic stability studies in carefully chosen test formulations. The evaluation of the melting temperature and the extent of aggregation of the API after a distinct time period of thermal stress resulted in a formulation buffer consisting of Histidine, Trehalose and Tween® 20. This buffer indicates stability and a long shelf-life of bispecific antibody NF-CUN297Q. The performed process- and preliminary formulation development are central prerequisites for GMP-compliant production. Bispecific antibody NF-CUN297Q produced with this GMP-process and formulated in the developed buffer will be suitable for application in a clinical phase I/IIa trial for the treatment of AML. This is an important prerequisite for the successful proof of safety and efficacy of this pharmaceutical drug.