Impact of fed-batch process intensification on the productivity and product quality of two CHO cell lines expressing unique novel molecular format proteins.

While monospecific antibodies have long been the foundational offering of protein therapeutics, recent advancements in antibody engineering have allowed for the development of far more complex antibody structures. Novel molecular format (NMF) proteins, such as bispecific antibodies (BsAbs), are structures capable of multispecific binding, allowing for expanded therapeutic functionality. As demand for NMF proteins continues to rise, biomanufacturers face the challenge of increasing bioreactor process productivity while simultaneously maintaining consistent product quality. This challenge is exacerbated when producing structurally complex proteins with asymmetric modalities, as seen in NMFs. In this study, the impact of a high inoculation density (HID) fed-batch process on the productivity and product quality attributes of two CHO cell lines expressing unique NMFs, a monospecific antibody with an Fc-fusion protein and a bispecific antibody, compared to low inoculation density (LID) platform fed-batch processes was evaluated. It was observed that an intensified platform fed-batch process increased product concentrations by 33 and 109% for the two uniquely structured complex proteins in a shorter culture duration while maintaining similar product quality attributes to traditional fed-batch processes.

An automated high inoculation density fed-batch bioreactor, enabled through N-1 perfusion, accommodates clonal diversity and doubles titers.

An important consideration for biopharmaceutical processes is the cost of goods (CoGs) of biotherapeutics manufacturing. CoGs can be reduced by dramatically increasing the productivity of the bioreactor process. In this study, we demonstrate that an intensified process which couples a perfused N-1 seed reactor and a fully automated high inoculation density (HID) N stage reactor substantially increases the bioreactor productivity as compared to a low inoculation density (LID) control fed-batch process. A panel of six CHOK1SV GS-KO® CHO cell lines expressing three different monoclonal antibodies was evaluated in this intensified process, achieving an average 85% titer increase and 132% space-time yield (STY) increase was demonstrated when comparing the 12-day HID process to a 15-day LID control process. These productivity increases were enabled by automated nutrient feeding in both the N-1 and N stage bioreactors using in-line process analytical technologies (PAT) and feedback control. The N-1 bioreactor utilized in-line capacitance to automatically feed the bioreactor based on a capacitance-specific perfusion rate (CapSPR). The N-stage bioreactor utilized in-line Raman spectroscopy to estimate real-time concentrations of glucose, phenylalanine, and methionine, which are held to target set points using automatic feed additions. These automated feeding methodologies were shown to be generalizable across six cell lines with diverse feed requirements. We show this new process can accommodate clonal diversity and reproducibly achieve substantial titer uplifts compared to traditional cell culture processes, thereby establishing a baseline technology platform upon which further increases bioreactor productivity and CoGs reduction can be achieved.