Continuous downstream process or connected batch process: Which one makes most sense for Biogen?

As biologics-based products move into therapeutic areas with large patient populations and high doses, batch processing may not be able to keep pace with product demands. At Biogen, we have been exploring a number of options that can enable higher productivity of our downstream processes. In addition to a fully “end to end” continuous process, a batch process comprised of several steps connected in series has been evaluated. In this presentation, technologies Biogen has evaluated to enable either continuous or connected processing will be shown. Multi-Column-Chromatography (MCC) for the Protein A capture chromatography step was evaluated in order to maximize resin utilization and increase productivity. Connecting subsequent polishing steps was explored to eliminate the need for large intermediate hold tanks. Various options for continuous diafiltration were assessed to enable a fully continuous UF/DF step. The results of these evaluations will be presented as well a comparison of the expected productivity and COGs for both process options

Process and cost modeling approaches for manufacturing operations utilizing multi- column chromatography applications

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Integration of upstream and downstream for a hybrid continuous process development and manufacturing for a stable monoclonal antibody produced in CHO cell culture

Process intensification by continuous operation has been successfully applied in the chemical industry, when batch processes matured several decades ago. Fully integrated upstream and downstream continuous processing has also shown great potential for increased productivity and reduced cost in biomanufacturing using mammalian cell culture. After a few decades of development, continuous or perfusion cell culture has demonstrated for manufacturing of labile proteins or low-titer processes. Due to significant challenges implementing fully integrated continuous biomanufacturing and the fact that fed-batch cell culture has not yet matured, fed-batch cell culture and batch chromatography steps are still predominant for stable protein manufacturing in the industry. In comparison to perfusion cell culture, continuous or semi-continuous downstream processing for stable monoclonal antibodies (mAbs) has developed within less than a decade. Due to a high titer, e.g., 8-10 g/L, already achieved via fed-batch cell culture, which challenges the processing capacity for batch downstream commercial manufacturing, the demand of continuous chromatography operation dramatically increases. Here, we present a case study developing a hybrid continuous upstream and downstream as our next generation process for production of a stable mAb. For upstream, we implemented N-1 perfusion seed, which significantly increased the seeding density for fed-batch production. After media and process parameter optimization, the product titer for the intensified fed-batch process with high-seed increased more than 100% over the original fed-batch process. It should be noted that the original fed-batch process was optimized and used for clinical manufacturing at 1000-L scale. For next generation downstream, we developed multi-column chromatography for Protein A step, automated VI step and integrated pool-less polishing chromatography steps with increased productivity and reduction in resin requirement, buffer consumption and processing time. The next generation process with perfusion N-1 seed and continuous chromatography steps has been scaled up in 500-L bioreactor, and now has been demonstrated for full implementation in a GMP manufacturing facility at the 2000-L scale. We will present full set of data to compare the original optimized batch process at 1000-L scale and the next generation process at 2000-L scale for the stable mAb production using CHO cell culture. We believe that the hybrid continuous process is relatively easy to develop and implement in GMP manufacturing with significantly higher productivity than conventional fed-batch process for now, while the hybrid continuous process lays a good foundation for us to further develop and implement fully continuous upstream and downstream process in manufacturing with even higher productivity in future

Next generation manufacturing for biologics: Integration of a hybrid model for continuous manufacturing concepts into a clinical facility

The “one size fits all” concept is rarely applicable in life, this is also true for the concept of continuous manufacturing where specific applications will differ based upon the requirements of the end user. This is the scenario we describe here in which aspects of continuous manufacturing for both upstream and downstream biologics manufacturing are being incorporated to address the current pipeline needs within Bristol-Myers Squibb. The application is for stable, easily expressed monoclonal antibody processes that require moderate volumes and throughputs, such as for most oncology or immuno-oncology therapies. However, this is countered by challenges of an expanding pipeline that necessitates looking beyond the current platform philosophy for how to modify the process with the goal to increase overall productivity in a flexible manner. BMS recently constructed a clinical biologics manufacturing facility on the Devens, Massachusetts campus with operations being initiated in two phases. The first phase start-up aligned with a traditional, but flexible (i.e., based upon disposable technologies) upstream and downstream processes and was rapidly brought on line. The second phase is the design and construction within that same manufacturing building purposely left unfinished to allow for the process development group to design and demonstrate a next generation concept for manufacturing. With respect to the upstream process, the decision was made to maintain a fed-batch production bioreactor philosophy, but to employ much higher inoculation densities through use of perfusion culture at the seed bioreactor stage generating the inoculum. This results in cultures with shorter durations and opportunities for increased titer. Selection of the overall cycle time is an optimization between cadence and bioreactor throughput. With respect to the downstream processes, numerous continuous manufacturing technologies were evaluated to handle the increased titers being generated in the bioreactors. These downstream technologies include continuous harvest technologies, multicolumn continuous chromatography for capture, integrated pool-less polishing steps, automated viral inactivation, single pass TFF and in-line diafiltration. The advantages for manufacturing cadence and overall throughput, as well as other outcomes including efforts to decrease perfusion media usage, and a significant reduction in downstream resin costs will be presented. Once the second phase is implemented, the facility will accommodate both traditional as well as this hybrid model for continuous manufacturing interchangeably. The overall benefit to support multiple clinical products and the higher titer/throughputs are expected to reduce the number of batches as well as eliminate resupply batches for clinical supply

Development of scalable semi-continuous downstream processes

The goal of this work is to establish an intensified downstream scheme for stable, high-titer monoclonal antibody (mAb) processes to achieve increased manufacturing productivity with short cadence, reduced cost, and small facility footprint. Several continuous manufacturing technologies including multi-column chromatography for capture, automated low-pH viral inactivation (low-pH VI), and integrated pool-less polishing steps were evaluated following consistent development methodologies for several mAbs. This presentation aims to provide an overview of the approaches to developing and integrating these discrete technologies in one cohesive process flow that fits manufacturing requirements in a flexible manner. Development efforts are illustrated in three major areas. First, twin-column continuous capture chromatography (CaptureSMB) was evaluated systematically for equivalency assessment comparing to traditional batch operation for different molecules. Development data showed overall comparable chromatography performance, while certain trends were found to be molecule/process specific. For executing viral clearance studies, scalable models were developed using CaptureSMB and a surrogate system employing standard batch chromatography with flow path modifications to mimic the loading strategy of CaptureSMB. We also introduce a model-assisted process characterization approach toward validation of continuous twin-column capture chromatography owing to increased process understanding. Second, experimental studies and computational fluid dynamics (CFD) modeling were used to reduce the risk of product aggregation in low-pH VI manufacturing operation. For various mixing systems, localized low-pH zones were characterized quantitatively to avoid the undesirable conditions that could cause severe aggregate formation during acid adjustment. The modeling tool integrated with mAb aggregation measurements facilitates the optimization of operating parameters (e.g., titrant addition rate, impeller agitation) and automation strategy to ensure robust VI scale-up performance. Third, various scenarios of integrated pool-less polishing steps operated in flowthrough-flowthrough (FT-FT) or flowthrough-bind/elute (FT-B/E) mode were evaluated with or without inline adjustment between the two steps. Performance and quality attributes are compared for integrated and decoupled polishing steps, with an example describing the development and optimization workflow for a specific mAb process. Finally, implication to process development timelines, scale-up performance and practical challenges to process implementation in the new 2000-L manufacturing facility will be discussed

Upstream control strategy development for afucosylated species in mAb biomanufacturing

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Role of the gradient slope during the product internal recycling for the multicolumn countercurrent solvent gradient purification of PEGylated proteins

Protein PEGylation, i.e. functionalization with poly(ethylene glycol) chains, has been demonstrated an ef-ficient way to improve the therapeutic index of these biopharmaceuticals. We demonstrated that Multi -column Countercurrent Solvent Gradient Purification (MCSGP) is an efficient process for the separation of PEGylated proteins (Kim et al., Ind. and Eng. Chem. Res. 2021, 60, 29, 10764-10776), thanks to the internal recycling of product-containing side fractions. This recycling phase plays a critical role in the economy of MCSGP as it avoids wasting valuable product, but at the same time impacts its productivity extending the overall process duration. In this study, our aim is to elucidate the role of the gradient slope within this recycling stage on the yield and productivity of MCSGP for two case-studies: PEGylated lysozyme and an industrially relevant PEGylated protein. While all the examples of MCSGP in the literature refer to a single gradient slope in the elution phase, for the first time we systematically investigate three differ-ent gradient configurations: i) a single gradient slope throughout the entire elution, ii) recycling with an increased gradient slope, to shed light on the competition between volume of the recycled fraction and required inline dilution and iii) an isocratic elution during the recycling phase. The dual gradient elution proved to be a valuable solution for boosting the recovery of high-value products, with the potential for alleviating the pressure on the upstream processing.(c) 2023 Elsevier B.V. All rights reserved

Design and economic investigation of a Multicolumn Countercurrent Solvent Gradient Purification unit for the separation of an industrially relevant PEGylated protein

Conjugation of biopharmaceuticals to polyethylene glycol chains, known as PEGylation, is nowadays an efficient and widely exploited strategy to improve critical properties of the active molecule, including stability, biodistribution profile, and reduced clearance. A crucial step in the manufacturing of PEGylated drugs is the purification. The reference process in industrial settings is single-column chromatography, which can meet the stringent purity requisites only at the expenses of poor product recoveries. A valuable solution to this trade-off is the Multicolumn Countercurrent Solvent Gradient Purification (MCSGP), which allows the internal and automated recycling of product-containing side fractions that are typically discarded in the batch processes. In this study, an ad hoc design procedure was applied to the single-column batch purification of an industrially relevant PEGylated protein, with the aim of defining optimal collection window, elution duration and elution buffer ionic strength to be then transferred to the MCSGP. This significantly alleviates the design of the continuous operation, subjected to manifold process parameters. The MCSGP designed by directly transferring the optimal parameters allowed to improve the yield and productivity by 8.2% and 17.8%, respectively, when compared to the corresponding optimized batch process, ensuring a purity specification of 98.0%. Once the efficacy of MCSGP was demonstrated, a detailed analysis of its cost of goods was performed and compared to the case of single-column purification. To the best of our knowledge, this is the first example of a detailed economic investigation of the MCSGP across different manufacturing scenarios and process cadences of industrial relevance, which demonstrated not only the viability of this continuous technology but also its flexibility