Protein A affinity membrane adsorbers are a promising alternative to resins to intensify the manufacturing of monoclonal antibodies. This study examined the process performance of convective diffusive membrane adsorbers operated in batch and continuous multi-column mode. Therefore, three different processes were compared regarding membrane utilization, productivity, and buffer consumption: the batch process, the rapid cycling parallel multi-column chromatography process, and the rapid cycling simulated moving bed process. The influence of the monoclonal antibody loading concentration (between 0.5 g L-1 and 5.2 g L-1) and the loading flow rate (between 1.25 MV min-1 and 10 MV min-1) on the monoclonal antibody binding behavior of the membrane adsorber were studied with breakthrough curve experiments. The determined breakthrough curves were used to calculate the monoclonal antibody dynamic binding capacity, the duration of the loading steps for each process, and the number of required membrane adsorbers for the continuous processes rapid cycling parallel multi-column chromatography and rapid cycling simulated moving bed. The highest productivity for the batch (176 g L-1 h-1) and rapid cycling parallel multi-column chromatography process (176 g L-1 h-1) was calculated for high monoclonal antibody loading concentrations and low loading flow rates. In contrast, the rapid cycling simulated moving bed process achieved the highest productivity (217 g L-1 h-1) for high monoclonal antibody loading concentrations and loading flow rates. Furthermore, due to the higher membrane utilization, the buffer consumption of the rapid cycling simulated moving bed process (1.1 L g-1) was up to 1.9 times lower than that of the batch or rapid cycling parallel multi-column chromatography operation (2.1 L g-1).

Harmonizing unit operations in the downstream process of monoclonal antibodies (mAbs) has a high potential to overcome throughput limitations and reduce manufacturing costs. This study proposes a streamlined clarification and capture (S-CC) process concept for the continuous processing of cell broth harvested from a connected bioreactor. The process was realized with a fluidized bed centrifuge connected to depth and sterile filters, a surge tank, and a multi-column chromatography (MCC) unit. The MCC unit was operated in the rapid cycling simulated moving bed (RC-BioSMB) mode with five convective diffusive membrane adsorbers (MAs). A control strategy and the surge tank were used to adjust the loading flow rate of the MCC unit. The mAb was recovered with a total process yield of 90%, with high removal of the process-related impurities HCP (2.1 LRV) and DNA (2.9 LRV). Moreover, the S-CC process productivity of 4.2 g h- 1 was up to 5.3 times higher than for comparable, hypothetical batch MA processes. In addition, the buffer consumption of the capture step could be reduced from 2.0 L g- 1 in batch mode to 1.2 L g- 1 in the RC-BioSMB mode. These results demonstrate the high potential of streamlined interconnected unit operations to improve the overall mAb downstream process performance.

The intensification of industrial biopharmaceutical production and the integration of process steps pave the way for patients to access affordable treatments. The predominantly batchwise biomanufacturing of established cell clarification technologies, stainless steel disc stack centrifugation (DSC) and single-use (SU) depth filtration (DF), pose technological and economical bottlenecks, that include low biomass loading capacities and low product recoveries. Therefore, a novel SU-based clarification platform was developed combining fluidized bed centrifugation (FBC) with integrated filtration. The feasibility of this approach was investigated for high cell concentration with more than 100E6 cells/mL. Furthermore, scalability to 200 L bioreactor scale was tested for moderate cell concentrations. In both trials, low harvest turbidities (4 NTU) and superior antibody recoveries (95%) were achieved. The impact on the overall economics of industrial SU biomanufacturing using an up-scaled FBC approach was compared with DSC and DF technologies for different process parameters. As a result, the FBC showed to be the most cost-effective alternative for annual mAb production below 500 kg. In addition, the FBC clarification of increasing cell concentrations was found to have minimal impact on overall process costs, in contrast to established technologies, demonstrating that the FBC approach is particularly suitable for intensified processes.

A flow-through process for monoclonal antibody (mAb) purification has been created by integrating two different membrane adsorbers (MAs) with single-pass tangential flow filtration (SPTFF) for continuous buffer exchange. First, a design of experiments (DoE) was carried out to investigate the influence of pH and conductivity on the removal of desoxyribonucleic acid (DNA) and host cell proteins (HCP), and to select buffer conditions for mAb flow-through mode operation for four different MAs. As next, breakthrough curve experiments were performed with the selected buffer conditions for each MA to analyze the binding behavior of mAb, DNA, and HCP. Then, the influence of the MA sequence in the double flow-through process on the mAb yield and DNA and HCP removal was studied in batch mode. The best sequence of MAs was used for the final integrated double flow-through polishing process, where the MAs were directly connected via SPTFF. The flow-through of the first (anion exchange) MA was continuously diafiltrated via SPTFF to buffer conditions for the second (cation exchange) MA. In this way, DNA (< 2 ppm) and HCP (< 29 ppm) were removed with simultaneously high mAb yield. Moreover, 36 times higher throughput was obtained than with a standard process that combines a packed-bed column with CEX resin in a bind and elute mode with an AEX MA in flow-through mode.

Monoclonal antibodies (mAb) are commonly manufactured by either discontinuous operations like fed-batch (FB) or continuous processes such as steady-state perfusion. Both process types comprise opposing advantages and disadvantages in areas such as plant utilization, feasible cell densities, media consumption and process monitoring effort. In this study, we show feasibility of a promising novel hybrid process strategy that combines beneficial attributes of both process formats. In detail, our strategy comprises a short duration FB, followed by a fast media exchange and cell density readjustment, marking the start of the next FB cycle. Utilizing a small-scale screening tool, we were able to identify beneficial process parameters, including FB interval duration and reinoculation cell density, that allow for multiple cycles of the outlined process in a reproducible manner. In addition, we could demonstrate scalability of the process to a 5L benchtop system, using a fluidized-bed centrifuge as scalable media exchange system. The novel process showed increased productivity (+217%) as well as longer cultivation duration, in comparison to a standard FB with a significantly lower media consumption per produced product (−50%) and a decreased need for process monitoring, in comparison to a perfusion cultivation. Further, the process revealed constant glycosylation pattern in comparison to the perfusion cultivation and has strong potential for further scale-up, due to the use of fully scalable cultivation and media exchange platforms. In summary, we have developed a novel hybrid process strategy that tackles the key challenges of current biomanufacturing of either low productivity or high media consumption, representing a new and innovative approach for future process intensification efforts.

Monoclonal antibodies are the workhorse of the pharmaceutical industry due to their potential to treat a variety of different diseases while providing high specificity and efficiency. As a consequence, a variety of production processes have been established within the biomanufacturing industry. However, the rapidly increasing demand for therapeutic molecules amid the recent COVID-19 pandemic demonstrated that there still is a clear need to establish novel, highly productive, and flexible production processes. Within this work, we designed a novel discontinuous process by combining two intensification strategies, thus increasing inoculation density and media exchange via a fluidized bed centrifuge, to fulfill the need for a flexible and highly productive production process for therapeutic molecules. To establish this new process, firstly, a small-scale experiment was conducted to verify synergies between both intensification strategies, followed by a process transfer towards the proof-of-concept scale. The combination of these two-process intensification measures revealed overall synergies resulting in decreased process duration (-37%) and strongly enhanced product formation (+116%) in comparison to the not-intensified standard operation. This led to an impressive threefold increase in space-time yield, while only negligible differences in product quality could be observed. Overall, this novel process not only increases the ways to react to emergency situations thanks to its flexibility and possible short development times, but also represents a possible alternative to the current established processes due to high increases in productivity, in comparison to standard fed-batch operations.

The semi and fully continuous production of monoclonal antibodies (mAbs) has been gaining traction as a lower cost, and efficient production of mAbs to broaden patient access. To be truly flexible and adaptive to process demands, the industry has lacked sufficient advanced control strategies. The variation of the upstream product concentration typically cannot be handled by the downstream capture step, which is configured for a constant feed concentration and fixed binding capacity. This inflexibility leads to losses of efficiency and product yield. This study shows that these challenges can be overcome by a novel advanced control strategy concept that includes dynamic control throughout a perfusion bioreactor, with cell retention by alternating tangential flow, integrated with simulated moving bed (SMB) multi-column chromatography. The automation workflow and advanced control strategy were implemented through the use of a visual programming development environment. This enabled dynamic flow control across the upstream and downstream process integrated with a dynamic column loading of the SMB. A sensor prototype, based on continuous biolayer interferometry measurements was applied to detect mAb breakthrough within the last column flow-through to manage column switching. This novel approach provided higher specificity and lower background signal compared to commonly used spectroscopy methods, resulting in an optimized resin utilization while simultaneously avoiding product loss. The dynamic loading was found to provide a twofold increase of the mAb concentration in the eluate compared to a conservative approach with a predefined recipe with similar impurity removal. This concept shows that advanced control strategies can lead to significant process efficiency and yield improvement.

Monoclonal antibodies (mAb) have gained enormous therapeutic application during the last decade as highly efficient and flexible tools for the treatment of various diseases. Despite this success, there remain opportunities to drive down the manufacturing costs of antibody-based therapies through cost efficiency measures. To reduce production costs, novel process intensification methods based on state-of-the-art fed-batch and perfusion have been implemented during the last few years. Building on process intensification, we demonstrate the feasibility and benefits of a novel, innovative hybrid process that combines the robustness of a fed-batch operation with the benefits of a complete media exchange enabled through a fluidized bed centrifuge (FBC). In an initial small-scale FBC-mimic screening, we investigated multiple process parameters, resulting in increased cell proliferation and an elongated viability profile. Consecutively, the most productive process scenario was transferred to the 5-L scale, further optimized and compared to a standard fed-batch process. Our data show that the novel hybrid process enables significantly higher peak cell densities (163%) and an impressive increase in mAb amount of approximately 254% while utilizing the same reactor size and process duration of the standard fed-batch operation. Furthermore, our data show comparable critical quality attributes (CQAs) between the processes and reveal scale-up possibilities and no need for extensive additional process monitoring. Therefore, this novel process intensification strategy yields strong potential for transfer into future industrial manufacturing processes.

Process intensification strives for more efficient conversion of raw materials into products while minimizing resource usage. In the biopharma industry, typical intensification gains include increased plant capacities, reduced raw material costs, smaller facilities and improved sustainability. Rapid growth combined with increasingly diverse and challenging molecule formats in the biopharma sector necessitate strategies for fast development of highly productive and cost-efficient processes. Using monoclonal antibody production in CHO cells as example, we present intensification techniques and process sequences that deliver synergistic benefits like increasing space-time yields up to 10-fold, shortening production runs by 30%, or saving numerous days in cell expansion. Combining highly productive perfusion with continuous downstream purification promises improved yields and production economics, especially for advanced, often labile molecules. Predictive scale-down models demonstrated here for selecting intensification-ready cell lines and for rapid setup of intensified processes are essential to realize such benefits broadly in the industry. They enable testing and quick tailoring of intensification alternatives like high-inoculation fed-batch or perfusion to suit molecule needs, facility space, and financial conditions. Since many techniques also readily extend to other protein biotherapeutics, intensification can be regarded as key pillar enabling fast, cost-efficient development and production of biopharma products in the coming years.

Precipitation and flocculation pretreatments promise improved clarification of cell culture fluids (CCF) to intensify the production of monoclonal antibodies (mAb). However, such pretreatments pose the risks to alter the mAb and damage cells. This can be additionally exacerbated by the subsequent clarification process, for example by high shear forces during disk stack centrifugation, resulting in a release of host cell impurities. To overcome these limitations and enhance the clarification particularly of cell cultures with low viability and thus a high level of impurities, this study investigated low-pH precipitation and cationic polymer flocculation, each in combination with a mild fluidized bed centrifuge (FBC) separation and a subsequent filtration step. Therefore, low-viable CCF´s were pretreated and characterized to investigate the effects of additives on CCF composition and stability. In clarification experiments, both pretreatments achieved similar FBC throughput compared to an untreated reference but increased the maximum filter throughput up to four times. Furthermore, high mAb recoveries (> 91%), low turbidities (< 3.1 NTU) and high DNA removals (> 91%) were achieved. Similar glycan profiles and dimer ratios suggest consistent mAb quality. These findings have a great potential to intensify mAb downstream processing with both CCF pretreatments using a FBC clarification approach.

The interest in extracellular vesicles (EVs) has been increased in recent years due to their potential application in diagnosis and therapy of severe diseases. The versatile fields of application due to the numerous possible cargos and the targeted delivery system make them a promising biopharmaceutical product. However, their broad size range as well as varied surface protein content result in challenges for the purification, characterization, and quantification. In this study a novel method, based on high-resolution flow cytometry, was examined for the enumeration of EVs in purified as well as crude process samples. In addition to quantification, samples were characterized by dynamic light scattering, zeta potential measurement, and analytical size exclusion chromatography. It has been demonstrated that EVs were successfully enumerated with the novel method, offering great benefits for development and monitoring of EV processes.

Improvements of the upstream process for the production of monoclonal antibodies (mAb) has resulted in mammalian cell cultures with significantly increased cell densities. These high cell density (HCD) broths pose severe challenges for established downstream unit operations due to the increased biomass load. To meet these challenges, aqueous two-phase extraction was examined as a potential method for the clarification, as well as first stage purification of a Chinese hamster ovary expressed mAb with cell densities up to 70 center dot 10(6)cells mL(-1). A clarification with 99 % yield of mAb and 68 % DNA removal was accomplished, which contributes to enable a foundation to the industrial application of aqueous two-phase extraction for manufacturing of mAb from HCD cultivations.

Intensified high cell density (HCD) processes for the production of therapeutic proteins have already reached cell concentrations up to 100 million cells/mL (> 300 g/L wet cell weight, WCW). The clarification of such HCD processes using technologies established for conventional fed-batch processes (< 100 g/L WCW) is faced with low biomass loading capacities and relatively low product recoveries, which limit their applicability. This and the growing demand for single-use (SU) technologies increases the pressure to develop scalable, robust and cost-effective SU based HCD clarification solutions. Therefore, in this study a SU fluidized bed centrifuge (FBC) system was investigated to clarify HCD broths with 100 million cells/mL from a monoclonal antibody production process. FBC parameters including process volumes and flow rates during the FBC operating steps were optimized so that an almost complete removal of biomass and product recoveries of 95 % were achieved. In addition, the mild centrifugation conditions were demonstrated by the superior cell viability for the separated cells while minimizing release of host cell impurities. Furthermore, a post-centrifugal filter screening showed acceptable turbidities below 5 NTU of the sterile filtrate. This concept of FBC with subsequent filtration offers excellent potential to achieve a robust HCD clarification process solution.

An alternative downstream process for the purification of a monoclonal antibody (mAb) based on aqueous two-phase extraction as clarification, capture and primary purification step was developed. For further purification unit operations which are commonly used for mAb platform processes were utilized. A diafiltration approach was used to combine virus inactivation and removal of phase forming components as well as low molecular weight impurities in one step followed by cation and anion exchange chromatography. Starting from cell containing cultivation broth, an overall mAb yield of 74 % was achieved within an application study. The process was optimized regarding mAb yield and the clearance of process related impurities like deoxyribonucleic acid as well as host cell proteins, which were removed to approximately 60 and 6000 ppm respectively. Critical product quality attributes, regarding glycosylation patterns, were also examined and remained unimpaired after the aqueous two-phase extraction. The alternative downstream process presented in this study offers great potential to improve mAb manufacturing.

Therapeutic monoclonal antibodies (mAb) are used for the treatment of numerous serious diseases, which have led to an increasing demand over the last decades. Increased cell density and mAb titer of the cultivation broth lead to great challenges for the subsequent clarification and capture operations in the downstream process. As an alternative approach to the conventional downstream process, a selective mAb extraction via an aqueous two-phase system (ATPS) directly from the cultivation broth of a mAb producing industrial relevant chinese hamster ovary (CHO) cell line was investigated. An efficient purification of the mAb was accomplished by the ATPS composition. The phase separation was realized by a newly developed membrane based phase separator. Moreover, a complete cell removal was integrated into this process by the used membrane. A selectivity between both phases was achieved by membrane modification. Yields up to 93% in the light phase and removal of process related impurities were obtained after aqueous two-phase extraction (ATPE). Phase separation performance as well as contact angles on the membrane were characterized for different ATPS. ATPE directly from the cultivation broth in combination with the new membrane based phase separation led to a mAb yield of 78% with a simultaneous reduction of deoxyribonucleic acid (DNA) and host cell protein (HCP) load.