Evaluating end-to-end continuous antibody manufacture with column-free capture alternatives from economic, environmental, and robustness perspectives.

Process intensification efforts have renewed interest in the potential of end-to-end continuous manufacture with column-free capture alternatives. This article describes a decisional tool that encompasses mass balance and design equations, process economics, stochastic simulation and multi-criteria decision-making and enables the evaluation of different batch, and continuous flowsheets for monoclonal antibody (mAb) manufacture. The traditional batch process was compared with end-to-end continuous bioprocesses with either protein A capture or column-free capture employing aqueous two-phase extraction or precipitation from economic, environmental, and robustness perspectives. The cost of goods analysis predicted that continuous flowsheets could offer substantial cost savings (~20%-40%) over the batch process at low and medium annual commercial demands (100-500 kg); however, at tonnage demands they resulted in either comparable or higher costs. Comparing the continuous options, the continuous flowsheets with protein A or precipitation yielded similar COG/g values, while aqueous two-phase extraction presented higher costs. The analysis of overall process mass intensities accounting for water and consumables suggested that the continuous flowsheet with protein A would result in the lowest environmental burden. When the economic, environmental, and operational criteria were reconciled using multi-criteria decision-making analysis, the continuous protein A-based flowsheet was found to be the most favorable. A target analysis highlighted the need for process improvements in the following parameters to reduce the manufacturing costs of the continuous column-free capture options below that of protein A: the perfusion volumetric productivity, the harvested cell culture fluid percentage in column-free operations, the column-free step yields along with the implementation of buffer concentrates.

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.

Establishment of a novel cell line, CHO-MK, derived from Chinese hamster ovary tissues for biologics manufacturing.

Chinese hamster ovary (CHO) cells are widely used as a host for producing recombinant therapeutic proteins due to advantages such as human-like post-translational modification, correct protein folding, higher productivity, and a proven track record in biopharmaceutical development. Much effort has been made to improve the process of recombinant protein production, in terms of its yield and productivity, using conventional CHO cell lines. However, to the best of our knowledge, no attempts have been made to acquire new CHO cell lines from Chinese hamster ovary. In this study, we established and characterized a novel CHO cell line, named CHO-MK, derived from freshly isolated Chinese hamster ovary tissues. Some immortalized cell lines were established via sub-culture derived from primary culture, one of which was selected for further development toward a unique expression system design. After adapting serum-free and suspension culture conditions, the resulting cell line exhibited a considerably shorter doubling time (approximately 10 h) than conventional CHO cell lines (approximately 20 h). Model monoclonal antibody (IgG1)-producing cells were generated, and the IgG1 concentration of fed-batch culture reached approximately 5 g/L on day 8 in a 200-L bioreactor. The cell bank of CHO-MK cells was prepared as a new host and assessed for contamination by adventitious agents, with the results indicating that it was free from any such contaminants, including infectious viruses. Taking these findings together, this study showed the potential of CHO-MK cells with a shorter doubling time/process time and enhanced productivity in biologics manufacturing.

Engineering death resistance in CHO cells for improved perfusion culture.

The reliable and cost-efficient manufacturing of monoclonal antibodies (mAbs) is essential to fulfil their ever-growing demand. Cell death in bioreactors reduces productivity and product quality, and is largely attributed to apoptosis. In perfusion bioreactors, this leads to the necessity of a bleed stream, which negatively affects the overall process economy. To combat this limitation, death-resistant Chinese hamster ovary cell lines were developed by simultaneously knocking out the apoptosis effector proteins Bak1, Bax, and Bok with CRISPR technology. These cell lines were cultured in fed-batch and perfusion bioreactors and compared to an unmodified control cell line. In fed-batch, the death-resistant cell lines showed higher cell densities and longer culture durations, lasting nearly a month under standard culture conditions. In perfusion, the death-resistant cell lines showed slower drops in viability and displayed an arrest in cell division after which cell size increased instead. Pertinently, the death-resistant cell lines demonstrated the ability to be cultured for several weeks without bleed, and achieved similar volumetric productivities at lower cell densities than that of the control cell line. Perfusion culture reduced fragmentation of the mAb produced, and the death-resistant cell lines showed increased glycosylation in the light chain in both bioreactor modes. These data demonstrate that rationally engineered death-resistant cell lines are ideal for mAb production in perfusion culture, negating the need to bleed the bioreactor whilst maintaining product quantity and quality.

Choices of capture chromatography technology in antibody manufacturing processes.

The capture process employed in monoclonal antibody downstream purification is not only the most critically impacted process by increased antibody titer resulting from optimized mammalian cell culture expression systems, but also the most important purification step in determining overall process throughput, product quality, and economics. Advances in separation technology for capturing antibodies from complex feedstocks have been one focus of downstream purification process innovation for past 10 years. In this study, we evaluated new generation chromatography resins used in the antibody capture process including Protein A, cation exchange, and mixed mode chromatography to address the benefits and unique challenges posed by each chromatography approach. Our results demonstrate the benefit of improved binding capacity of new generation Protein A resins, address the concern of high concentration surge caused aggregation when using new generation cation exchange resins with over 100mg/mL binding capacity, and highlight the potential of multimodal cation exchange resins for capture process design. The new landscape of capture chromatography technologies provides options to achieve overall downstream purification outcome with high product quality and process efficiency.