Process intensification has shown great potential to increase productivity and reduce costs in biomanufacturing. This case study describes the evolution of a manufacturing process from a conventional processing scheme at 1000-L scale (Process A, n=5) to intensified processing schemes at both 1000-L (Process B, n=8) and 2000-L scales (Process C, n=3) for the production of a monoclonal antibody by a Chinese hamster ovary cell line. For the upstream part of the process, we implemented an intensified seed culture scheme to enhance cell densities at the seed culture step (N-1) prior to the production bioreactor (N) by using either enriched N-1 seed culture medium for Process B or by operating the N-1 step in perfusion mode for Process C. The increased final cell densities at the N-1 step allowed for much higher inoculation densities in the production bioreactor operated in fed-batch mode and substantially increased titers by 4-fold from Process A to B and 8-fold from Process A to C, while maintaining comparable final product quality. Multiple changes were made to intensify the downstream process to accommodate the increased titers. New high-capacity resins were implemented for the Protein A and anion exchange chromatography (AEX) steps, and the cation exchange chromatography (CEX) step was changed from bind-elute to flow-through mode for the streamlined Process B. Multi-column chromatography was developed for Protein A capture, and an integrated AEX-CEX pool-less polishing steps allowed semi-continuous Process C with increased productivity as well as reductions in resin requirements, buffer consumption and processing times. A cost-of-goods analysis on consumables showed 6.7-10.1 fold cost reduction from the conventional Process A to the intensified Process C. The hybrid intensified process described here is easy to implement in manufacturing and lays a good foundation to develop a fully continuous manufacturing with even higher productivity in the future.

Although process intensification by continuous operation has been successfully applied in the chemical industry, the biopharmaceutical industry primarily uses fed-batch, rather than continuous or perfusion methods, to produce stable monoclonal antibodies (mAbs) from Chinese hamster ovary (CHO) cells. Conventional fed-batch bioreactors may start with an inoculation viable cell density (VCD) of ~0.5 × 10⁶ cells/mL. Increasing the inoculation VCD in the fed-batch production bioreactor (referred to as N stage bioreactor) to 2–10 × 10⁶ cells/mL by introducing perfusion operation or process intensification at the seed step (N-1 step) prior to the production bioreactor has recently been used because it increases manufacturing output by shortening cell culture production duration. In this study, we report that increasing the inoculation VCD significantly improved the final titer in fed-batch production within the same 14-day duration for 3 mAbs produced by 3 CHO GS cell lines. We also report that other non-perfusion methods at the N-1 step using either fed batch or batch mode with enriched culture medium can similarly achieve high N-1 final VCD of 22–34 × 10⁶ cells/mL. These non-perfusion N-1 seeds supported inoculation of subsequent production fed-batch production bioreactors at increased inoculation VCD of 3–6 × 10⁶ cells/mL, where these achieved titer and product quality attributes comparable to those inoculated using the perfusion N-1 seeds demonstrated in both 5-L bioreactors, as well as scaled up to 500-L and 1000-L N-stage bioreactors. To operate the N-1 step using batch mode, enrichment of the basal medium was critical at both the N-1 and subsequent intensified fed-batch production steps. The non-perfusion N-1 methodologies reported here are much simpler alternatives in operation for process development, process characterization, and large-scale commercial manufacturing compared to perfusion N-1 seeds that require perfusion equipment, as well as preparation and storage vessels to accommodate large volumes of perfusion media. Although only 3 stable mAbs produced by CHO cell cultures are used in this study, the basic principles of the non-perfusion N-1 seed strategies for shortening seed train and production culture duration or improving titer should be applicable to other protein production by different mammalian cells and other hosts at any scale biologics facilities.

Temperature shift (TS) to a hypothermic condition has been widely used during protein production processes that use Chinese hamster ovary (CHO) cells. The effect of temperature on cell growth, metabolites, protein titer and quality depends on cell line, product, and other bioreactor conditions. Due to the large numbers of experiments, which typically last 2–3 weeks each, limited systematic TS studies have been reported with multiple shift temperatures and steps at different times. Here, we systematically studied the effect of temperature on cell culture performance for the production of two monoclonal antibodies by industrial GS and DG44 CHO cell lines. Three 2–8 day short-duration methods were developed and validated for researching the effect of many different temperatures on CHO cell culture and quality attributes. We found that minor temperature differences (1–1.5 °C) affected cell culture performance. The kinetic parameters extracted from the short duration data were subsequently used to compute and predict cell culture performance in extended duration of 10–14 days with multiple TS conditions for both CHO cell lines. These short-duration culture methods with kinetic modeling tools may be used for effective TS optimization to achieve the best profiles for cell growth, metabolites, titer and quality attributes. Although only three short-duration methods were developed with two CHO cell lines, similar short-duration methods with kinetic modeling may be applied for different hosts, including both microbial and other mammalian cells.

The goal of cell culture process intensification is to increase volumetric productivity, generally by increasing viable cell density (VCD), cell specific productivity or production bioreactor utilization in manufacturing. In our previous study, process intensification in fed-batch production with higher titer or shorter duration was demonstrated by increasing the inoculation seeding density (SD) from ~ 0.6 (Process A) to 3–6 × 10⁶ cells/mL (Process B) in combination with media enrichment. In this study, we further increased SD to 10–20 × 10⁶ cells/mL (Process C) using perfusion N-1 seed cultures, which increased titers already at industrially relevant levels by 100% in 10–14 day bioreactor durations for four different mAb-expressing CHO cell lines. Redesigned basal and feed media were critical for maintaining higher VCD and cell specific productivity during the entire production duration, while medium enrichment, feeding strategies and temperature shift optimization to accommodate high VCDs were also important. The intensified Process C was successfully scaled up in 500-L bioreactors for 3 of the 4 mAbs, and quality attributes were similar to the corresponding Process A or Process B at 1000-L scale. The fed-batch process intensification strategies developed in this study could be applied for manufacturing of other mAbs using CHO and other host cells.

During biopharmaceutical process development, it is important to improve titer to reduce drug manufacturing costs and to deliver comparable quality attributes of therapeutic proteins, which helps to ensure patient safety and efficacy. We previously reported that relative high-iron concentrations in media increased titer, but caused unacceptable coloration of a fusion protein during early-phase process development. Ultimately, the fusion protein with acceptable color was manufactured using low-iron media, but the titer decreased significantly in the low-iron process. Here, long-term passaging in low-iron media is shown to significantly improve titer while maintaining acceptable coloration during late-phase process development. However, the long-term passaging also caused a change in the protein charge variant profile by significantly increasing basic variants. Thus, we systematically studied the effect of media components, seed culture conditions, and downstream processing on productivity and quality attributes. We found that removing β-glycerol phosphate (BGP) from basal media reduced basic variants without affecting titer. Our goals for late-phase process development, improving titer and matching quality attributes to the early-phase process, were thus achieved by prolonging seed culture age and removing BGP. This process was also successfully scaled up in 500-L bioreactors. In addition, we demonstrated that higher concentrations of reactive oxygen species were present in the high-iron Chinese hamster ovary cell cultures compared to that in the low-iron cultures, suggesting a possible mechanism for the drug substance coloration caused by high-iron media. Finally, hypotheses for the mechanisms of titer improvement by both high-iron and long-term culture are discussed.