Process Development and Manufacturing Considerations for Multispecific (Bispecific and Trispecific) Antibodies: Case Study

Antibodies as a therapeutic treatment have been the focus of numerous companies for many years, resulting in over 100 currently on the market. This has led to the creation of a rich understanding of how to develop and manufacture these molecules. The result has been the creation of platforms consisting of high productivity cell lines and optimized culture conditions that can generate titers as high as 10 g/L. These platforms have also seen the introduction of streamlined downstream processes which typically consist of two to three chromatography steps. In recent years, the bispecific and trispecific (or multispecific) antibodies have become a large area of development for most companies, as the ability to bind two different antigens at a time opens new and unique therapeutic areas. Although these are antibody-like, the complexity created due to the structurally diverse molecular formats and engineering adjustments presents a challenge to the current antibody development and manufacturing paradigm. New steps such as reactions to bring these molecules together as well as new impurities and stability issues have meant that these platforms have needed to be adjusted to enable production of suitable quantities of high-quality product. Expression and production of multispecific antibodies have brought new challenges and considerations to cell line generation. Depending upon molecular format, a strategy needs to be implemented encompassing choice of cell host organism (microbial vs. mammalian), and in the case of mammalian expression, number of cell lines generated (single vs. dual cell lines), as well as random versus targeted integration of transgenes. These initial choices can have far reaching implications, often necessitating expanded cell line screening efforts when compared to traditional monoclonals. Once a suitable cell line is created, the production of multispecific molecules uses culture conditions similar to typical antibodies. Some adjustments are required however as the engineering of these molecules may result in a higher occurrence of clip species formation or presence of different molecule fragments or impurities. Attention has been paid to ways to reduce these impurities while increasing titers through culture growth conditions and setpoints. After production there are several considerations that must be focused on to achieve a final high-quality product. These complex molecules can be less stable than traditional antibodies and they tend to contain new and unique impurities that must be removed. Due to the engineering of the molecules, there can be issues with stability that may prevent traditional operations from being conducted such as low pH viral inactivation. This results in the need to design or find additional means to achieve sufficient and robust viral safety. Also, most of the molecules have some level of homodimers present, and due to the similarity of these impurities to the heterodimer, the separation and removal can be difficult. This often results in either the addition of new steps or the use of less traditional steps being employed.