Intensified fed‐batch (IFB), a popular cell culture intensification strategy, has been widely used for productivity improvement through high density inoculation followed by fed‐batch cultivation. ...However, such an intensification strategy may counterproductively induce rapidly progressing cell apoptosis and difficult‐to‐sustain productivity. To improve culture performance, we developed a novel cell culture process intermittent‐perfusion fed‐batch (IPFB) which incorporates one single or multiple cycles of intermittent perfusion during an IFB process for better sustained cellular and metabolic behaviors and notably improved productivity. Unlike continuous perfusion or other semi‐continuous processes such as hybrid perfusion fed‐batch with only early‐stage perfusion, IPFB applies limited times of intermittent perfusion in the mid‐to‐late stage of production and still inherits bolus feedings on nonperfusion days as in a fed‐batch culture. Compared to IFB, an average titer increase of ~45% was obtained in eight recombinant CHO cell lines studied. Beyond IPFB, ultra‐intensified IPFB (UI‐IPFB) was designed with a markedly elevated seeding density of 20–80 × 106 cell/mL, achieved through the conventional alternating tangential flow filtration (ATF) perfusion expansion followed with a cell culture concentration step using the same ATF system. With UI‐IPFB, up to ~6 folds of traditional fed‐batch and ~3 folds of IFB productivity were achieved. Furthermore, the application grounded in these two novel processes showed broad‐based feasibility in multiple cell lines and products of interest, and was proven to be effective in cost of goods reduction and readily scalable to a larger scale in existing facilities.
Currently, the mammalian biomanufacturing industry explores process intensification (PI) to meet upcoming demands of biotherapeutics while keeping production flexible but, more importantly, as ...economic as possible. However, intensified processes often require more development time compared with conventional fed‐batches (FBs) preventing their implementation. Hence, rapid and efficient, yet straightforward strategies for PI are needed. In this study we demonstrate such a strategy for the intensification of an N‐stage FB by implementing N‐1 perfusion cell culture and high inoculum cell densities resulting in a robust intensified FB (iFB). Furthermore, we show successful combination of such an iFB with the addition of productivity enhancers, which has not been reported so far. The conventional CHO cell FB process was step‐wise improved and intensified rapidly in multi‐parallel small‐scale bioreactors using N‐1 perfusion. The iFBs were performed in 15 and 250 ml bioreactors and allowed to evaluate the impact on key process indicators (KPI): the space–time yield (STY) was successfully doubled from 0.28 to 0.55 g/L d, while product quality was maintained. This gain was generated by initially increasing the inoculation density, thus shrinking process time, and second supplementation with butyric acid (BA), which reduced cell growth and enhanced cell‐specific productivity from ~25 to 37 pg/(cell d). Potential impacts of PI on cell metabolism were evaluated using flux balance analysis. Initial metabolic differences between the standard and intensified process were observed but disappeared quickly. This shows that PI can be achieved rapidly for new as well as existing processes without introducing sustained changes in cellular and metabolic behavior.
Process intensification is increasingly used in the mammalian biomanufacturing industry. The key driver of this trend is the need for more efficient and flexible production strategies to cope with ...the increased demand for biotherapeutics predicted in the next years. Therefore, such intensified production strategies should be designed, established, and characterized. We established a CHO cell process consisting of an intensified fed‐batch (iFB), which is inoculated by an N‐1 perfusion process that reaches high cell concentrations (100 × 106 c ml−1). We investigated the impact of butyric acid (BA) supplementation in this iFB process. Most prominently, higher cellular productivities of more than 33% were achieved, thus 3.5 g L−1 of immunoglobulin G (IgG) was produced in 6.5 days. Impacts on critical product quality attributes were small. To understand the biological mechanisms of BA in the iFB process, we performed a detailed transcriptomic analysis. Affected gene sets reflected concurrent inhibition of cell proliferation and impact on histone modification. These translate into subsequently enhanced mechanisms of protein biosynthesis: enriched regulation of transcription, messenger RNA processing and transport, ribosomal translation, and cellular trafficking of IgG intermediates. Furthermore, we identified mutual tackling points for optimization by gene engineering. The presented strategy can contribute to meet future requirements in the continuously demanding field of biotherapeutics production.
High demand in manufactured biologics drives the continued need for increased productivity. In this study elevated lactate metabolization resulted in improved metabolic efficiency and cellular ...productivity for a readily intensified high titer fed‐batch process. Scheduled base or lactate feeds during the stationary growth phase led to increased titers (+9% and +8% respectively) without impacting the overall growth performance. The higher lactate consumption induced by either feed strategy substituted for glutamate catabolism and consequently reduced ammonia build‐up. Direct correlation between increased titers and reduced ammonia levels was shown. Product quality attributes were impacted by both feeding strategies but could be matched with the control process by shortening the cell culture duration while maintaining titer constant.
In the field of therapeutic antibody production, diversification of fed-batch strategies is flourishing in response to the market demand. All manufacturing approaches tend to follow the generally ...accepted dogma of increasing titer since it directly increases manufacturing output. While titer is influenced by the biomass (expressed as IVCD), the culture time and the cell-specific productivity (
q
P
), we changed independently each of these parameters to tune our process strategy towards adapted solutions to individual manufacturing needs. To do so, we worked separately on the increase of the IVCD as high seeding fed-batch capacity. Yet, as intensified fed-batch may not always be possible due to limited facility operational mode, we also separately increased the
q
P
with the addition of specific media additives. Both strategies improved titer by 100% in 14 days relative to the standard fed-batch process with moderate and acceptable changes in product quality attributes. Since intensified fed-batch could rival the cell-specific productivity of a conventional fed-batch, we developed novel hybrid strategies to either allow for acceptable seeding densities without compromising productivity, or alternatively, to push the productivity the furthest in order to reduce timelines.
The conventional fed-batch process characterized by a low titer currently challenges pharmaceutical development. Process optimization by applying a perfusion process in the pre-stage and subsequent ...production phase at a high seeding density (HSD) can meet this challenge. In this study, we employed a simplified approach based on measured experiments, namely segmented modeling, to systematically analyze an HSD fed-batch process compared to a standard process. A comparison indicated that the metabolic phases of HSD processes are not only shifted in time, but metabolite trends show an altered metabolism. In an extended study, we integrated the intracellular fluxes determined by a metabolic flux analysis into the segmented modeling approach. Compared to using only extracellular rates, similar phases are identified, and this highlights the reliability of phase identification modeling using extracellular rates only. Furthermore, the segmented linear regression approach is used to create a model that describes cellular behavior and that can be used to predict potential improvements in the feeding strategy and in harvest viability. Here, overfeeding was eliminated and a significantly higher titer was achieved. This work provides insights into the overall metabolic changes in the HSD process and paves the way towards the optimization of the feeding regime.
Due to their high specificity, monoclonal antibodies (mAbs) have garnered significant attention in recent decades, with advancements in production processes, such as high-seeding-density (HSD) ...strategies, contributing to improved titers. This study provides a thorough investigation of high seeding processes for mAb production in Chinese hamster ovary (CHO) cells, focused on identifying significant metabolites and their interactions. We observed high glycolytic fluxes, the depletion of asparagine, and a shift from lactate production to consumption. Using a metabolic network and flux analysis, we compared the standard fed-batch (STD FB) with HSD cultivations, exploring supplementary lactate and cysteine, and a bolus medium enriched with amino acids. We reconstructed a metabolic network and kinetic models based on the observations and explored the effects of different feeding strategies on CHO cell metabolism. Our findings revealed that the addition of a bolus medium (BM) containing asparagine improved final titers. However, increasing the asparagine concentration in the feed further prevented the lactate shift, indicating a need to find a balance between increased asparagine to counteract limitations and lower asparagine to preserve the shift in lactate metabolism.
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