Abstract Perfusion processing has established itself as a powerful intensification strategy for biopharmaceutical production processes of recombinant proteins. However, in the relatively young field of cell therapeutics manufacturing, this tool has been overlooked so far and with rising approvals, this field is in dire need of scalable and efficient production processes. In this regard, perfusion operation mode and related cell retention devices can be used to support upstream and downstream processing by automating operations, reducing contamination risk and ensuring stable cell quality. In this review, the history of cell retention technologies developed for suspension cell processing is laid out alongside the perfusion-based process intensification strategies they made possible. It is summarized how these technologies could be used to intensify the upstream processing of therapeutic adherent cells and to what degree perfusion processing of these demanding cells is already described in literature. Additionally, the applicability of cell retention devices for harvesting and downstream processing of therapeutic cells, which mainly consists of cell washing and formulation steps, is elaborated. In conclusion, through the implementation of scalable, single-use, good manufacturing practices compliant cultivation systems and cell retention devices, it should be possible substantially to accelerate the development of therapeutic adherent cell manufacturing strategies. Key points • Perfusion can automate medium exchanges in therapeutic adherent cell manufacturing • Perfusion technologies from suspension cell processing can be translated to adherent cell manufacturing • Scalable single-use perfusion systems are needed for the production of cell therapies

Whether for classic biologics or advanced therapy medicinal products (ATMPs), laboratory applications, small-scale or large-scale productions, single-use technology (SUT), and its associated consumables are used everywhere. The advantages of deploying SUTs are well known. Wherever a single-use solution is available, it is tested and ultimately used. Based on milestones in the development of single-use systems and single-use platform technologies, this article provides an up-to-date overview of products available on the market and their manufacturers/suppliers. It also discusses process examples with SUTs, design options and configurations of single-use facilities and Switzerland's pioneering role in the development and implementation of SUTs. In addition, the authors show that SUT has already been established beyond the biopharmaceutical sector.

The production of clinically relevant quantities of human mesenchymal stromal cells (hMSCs) requires scalable and intensified manufacturing processes. For this reason, the applicability of alternating tangential flow filtration (ATF) and tangential flow depth filtration (TFDF) based cell retention systems for hMSC expansion on microcarriers (MCs) in perfusion mode was assessed. The processes were conducted in stirred tank bioreactors at a scale of 1.8 L and compared with repeated-batch cultivations. In the perfusion and repeated-batch control cultivations, competitive viable cell concentrations of ≈2.9 · 106 cells mL-1 were reached within a cultivation period of 5-7 days, resulting in an expansion factor of 41-57. The main difference between the operation modi was the aggregation behavior of the MCs. While the median MC aggregate diameter in the repeated-batch cultivation reached 470 μm, the ATF cell retention device constrained aggregate size to a median diameter of 250 µm. In the TFDF cultivation, the shear forces in the recirculation loop stripped most of the hMSCs from the MCs, resulting in the formation of spheroids that continued to proliferate, albeit at a decreased rate. While perfusion operation did not lead to increased productivity in this proof-of-concept study, manual handling and therefore contamination risk were reduced by replacing the repeated-batch process's daily 80% medium exchanges with automated perfusion operation. Additionally, the ATF system was shown to be useful for medium removal and washing of the MCs prior to adding the harvesting solution, which is highly valuable for cultivations conducted at larger scales. While the feasibility of ATF based cell retention for MC expansion processes could be demonstrated, increased growth area to medium ratios, i.e., higher MC concentrations, still need to be investigated to leverage the full potential of the perfusion process mode.

The manufacturing of allogeneic cell therapeutics based on human-induced pluripotent stem cells (hiPSCs) holds considerable potential to revolutionize the accessibility and affordability of modern healthcare. However, achieving the cell yields necessary to ensure robust production hinges on identifying suitable and scalable single-use (SU) bioreactor systems. While specific stirred SU bioreactor types have demonstrated proficiency in supporting hiPSC expansion at L-scale, others, notably instrumented SU multiplate and fixed-bed bioreactors, remain relatively unexplored. By characterizing these bioreactors using both computational fluid dynamics and experimental bioengineering methods, operating ranges were identified for the Xpansion® 10 and Ascent™ 1 m2 bioreactors in which satisfactory hiPSC expansion under serum-free conditions was achieved. These operating ranges were shown not only to effectively limit cell exposure to wall shear stress but also facilitated sufficient oxygen transfer and mixing. Through their application, almost 5 × 109 viable cells could be produced within 5 days, achieving expansion factors of up to 35 without discernable impact on cell viability, identity, or differentiation potential.

The perfusion mode has become increasingly important in biopharmaceutical production in recent years. A bioreactor system used in many laboratories for the development of monoclonal antibodies (mAbs) production processes is the Sartorius’ Ambr. 250 system. Vessels designed for perfusion mode are only available for its high throughput version, while the modular version of the Ambr 250 is not designed for perfusion mode. In this study, perfusion processes for the production of a mAb with Chinese Hamster Ovary (CHO) cells were realized in the Ambr 250 Modular in combination with Repligen’s ATF 1 single-use device for the first time, to the authors’ knowledge. After testing a semi-perfusion setup in well plates and the Ambr 250, an N−1 perfusion process was developed to produce ultra-high cell densities of more than 150 Å~ 106 cells mL−1 for the inoculation of subsequent mAb production processes. In a second step, continuous mAb production was successfully realized over 23 days in a proof-of-concept experiment, achieving a volumetric productivity of 0.65 g L−1 d−1. The results of the N−1 and continuous perfusion processes were comparable to a 3 L HyPerformaTM Glass bioreactor (Thermo Scientific) with an ATF 2 (Repligen).

Abstract To fully utilize the potential of human induced pluripotent stem cells (hiPSCs) for allogeneic stem cell–based therapies, efficient and scalable expansion procedures must be developed. For other adherent human cell types, the combination of microcarriers (MCs) and stirred tank bioreactors has been shown to meet these demands. In this study, a hiPSC quasi-perfusion expansion procedure based on MCs was developed at 100-mL scale in spinner flasks. Process development began by assessing various medium exchange strategies and MC coatings, indicating that the hiPSCs tolerated the gradual exchange of medium well when cultivated on Synthemax II–coated MCs. This procedure was therefore scaled-up to the 1.3-L Eppendorf BioBLU 1c stirred tank bioreactor by applying the lower limit of Zwietering’s suspension criterion ( $${N}_{s1u}$$ N s 1 u ), thereby demonstrating proof-of-concept when used in combination with hiPSCs for the first time. To better understand the bioreactor and its bioengineering characteristics, computational fluid dynamics and bioengineering investigations were performed prior to hiPSC cultivation. In this manner, improved process understanding allowed an expansion factor of ≈ 26 to be achieved, yielding more than 3 × 109 cells within 5 days. Further quality analyses confirmed that the hiPSCs maintained their viability, identity, and differentiation potential throughout cultivation. Key points • $${N}_{s1u}$$ N s 1 u can be used as a scale-up criterion for hiPSC cultivations in MC-operated stirred bioreactors • Uniform distribution and attachment of cells to the MCs are crucial for efficient expansion • Perfusion is advantageous and supports the cultivation of hiPSCs

The perfusion mode has become increasingly important in biopharmaceutical production in recent years. A bioreactor system used in many laboratories for the development of monoclonal antibodies (mAbs) production processes is the Sartorius’ Ambr. 250 system. Vessels designed for perfusion mode are only available for its high throughput version, while the modular version of the Ambr 250 is not designed for perfusion mode. In this study, perfusion processes for the production of a mAb with Chinese Hamster Ovary (CHO) cells were realized in the Ambr 250 Modular in combination with Repligen’s ATF 1 single-use device for the first time, to the authors’ knowledge. After testing a semi-perfusion setup in well plates and the Ambr 250, an N−1 perfusion process was developed to produce ultra-high cell densities of more than 150 Å~ 106 cells mL−1 for the inoculation of subsequent mAb production processes. In a second step, continuous mAb production was successfully realized over 23 days in a proof-of-concept experiment, achieving a volumetric productivity of 0.65 g L−1 d−1. The results of the N−1 and continuous perfusion processes were comparable to a 3 L HyPerformaTM Glass bioreactor (Thermo Scientific) with an ATF 2 (Repligen).

There are several ways to make antibody production processes more efficient and less expensive through process intensification. In this study, an ultra-high cell density working cell bank was used to inoculate production bioreactors directly with cells from cryovials. After lab-scale experiments, a fed-batch pilot-scale experiment was successfully conducted as a proof-of-concept. In addition, cells were cultivated in perfusion mode at 2 L scale. The intensification approach presented here allows the complete elimination of the inoculum production outside the production bioreactor and the creation of additional production capacity by saving time and space. In perfusion mode, an increase in bioreactor productivity of over 180% can be achieved compared to fed-batch mode.

The bioreactor is the centerpiece of the upstream processing in any biotechnological production process. Its design, the cultivation parameters, the production cell line, and the culture medium all have a major influence on the efficiency of the process and the result of the cultivation. Disposable bioreactors have been used for the past 20 years, playing a major role in process development and commercial production of high-value substances at medium scales.Our review deals with scalable, disposable bioreactors that have proven to be useful for the cultivation of plant cell and tissue cultures. Based on the definitions of terms and a categorization approach, the most commonly used, commercially available, disposable bioreactor types are presented below. The focus is on wave-mixed, stirred, and orbitally shaken bioreactors. In addition to their instrumentation and bioengineering characteristics, cultivation results are discussed, and emerging trends for the development of disposable bioreactors for plant cell and tissue cultures are also addressed.

Given the demands human induced pluripotent stem cell (hiPSC)-based therapeutics place on manufacturing, process intensification strategies which rapidly ensure the desired cell quality and quantity should be considered. Within the context of antibody and vaccine manufacturing, one-step inoculation has emerged as an effective strategy for intensifying the upstream process. This study therefore evaluated whether this approach could be applied to the expansion of hiPSCs in flasks under static and in microcarrier-operated stirred bioreactors under dynamic conditions. Our findings demonstrated that high density working cell banks containing hiPSCs at concentrations of up to 100 × 106 cells mL−1 in CryoStor® CS10 did not impair cell growth and quality upon thawing. Furthermore, while cell distribution, growth, and viability were comparable to routinely passaged hiPSCs, those subjected to one-step inoculation and expansion on microcarriers under stirred conditions were characterized by improved attachment efficiency (≈50%) following the first day of cultivation. Accordingly, the process development outlined in this study establishes the foundation for the implementation of this intensified approach at L-scale.

Modern production processes for biopharmaceuticals often work with very high cell densities. Moreover, there is a trend towards moving from fed-batch to continuous perfusion processes; a development that is influencing the requirements for bioreactor design and process control. In this study, the transfer of fed-batch and perfusion experiments between different cylindrical stirred lab-scale bioreactors and Thermo Scientific’sTM (Waltham, MA, USA) cubical HyPerformaTM DynaDriveTM Single-Use Bioreactor was investigated. Different scaling parameters were used, which were selected based on the requirements of the respective processes. Peak cell densities of up to 49 × 106 cells mL−1 and antibody titers of up to 5.2 g L−1 were achieved in 15- to 16-day fed-batch experiments. In 50-day perfusion cultivations, a viable cell volume of >100 mm3 mL−1 was maintained and more than 1 g L−1 d−1 of antibodies were harvested. The perfusion processes were automated with both cell bleed control and glucose concentration control. Cell retention was performed using Repligen’s (Waltham, MA, USA) XCell® ATF perfusion systems and single-use devices. In summary, approaches for successfully scaling highly productive fed-batch and perfusion processes between geometrically dissimilar lab and pilot scale bioreactors were demonstrated. The advantages of perfusion in comparison to fed-batch processes were also observed.

HEK293 is a widely used cell line in the fields of research and industry. It is assumed that these cells are sensitive to hydrodynamic stress. The aim of this research was to use particle image velocimetry validated computational fluid dynamics (CFD) to determine the hydrodynamic stress in both shake flasks, with and without baffles, and in stirred Minifors 2 bioreactors to evaluate its effect on the growth and aggregate size distribution of HEK293 suspension cells. The HEK FreeStyleTM 293-F cell line was cultivated in batch mode at different specific power inputs (from 63 W m-3 to 451 W m-3), whereby ≈60 W m-3 corresponds to the upper limit, which is what has been typically described in published experiments. In addition to the specific growth rate and maximum viable cell density VCDmax, the cell size distribution over time and cluster size distribution were investigated. The VCDmax of (5.77±0.02)·106cellsmL-1 was reached at a specific power input of 233 W m-3 and was 23.8% higher than the value obtained at 63 W m-3 and 7.2% higher than the value obtained at 451 W m-3. No significant change in the cell size distribution could be measured in the investigated range. It was shown that the cell cluster size distribution follows a strict geometric distribution whose free parameter p is linearly dependent on the mean Kolmogorov length scale. Based on the performed experiments, it has been shown that by using CFD-characterised bioreactors, the VCDmax can be increased and the cell aggregate rate can be precisely controlled.

The first part of this series on characterisation of bioreactors in the biopharmaceutical industry using computational fluid dynamics presented a literature review to illustrate how characterisation can be performed and which process engineering parameters can be determined using computational fluid dynamics (CFD). In addition, experimental validation methods were presented, and an overview of typical hardware and software was also provided. In this second part, a selection of the authors’ research results will be used to demonstrate how the process characterisation of mechanically driven bioreactors for the biopharmaceutical industry can be determined with CFD and then experimentally validated. Three stirred tank bioreactors with different filling volumes and stirrers were used to demonstrate power input and oxygen transfer in single- and two-phase simulations. For wave-mixed and orbitally shaken systems, the fluid flow was transiently simulated and experimentally validated. In addition, the power input was also determined for both systems.

Stem cell-based cell therapeutics and especially those based on human mesenchymal stem cells (hMSCs) and induced pluripotent stem cells (hiPSCs) are said to have enormous developmental potential in the coming years. Their applications range from the treatment of orthopedic disorders and cardiovascular diseases to autoimmune diseases and even cancer. However, while more than 27 hMSC-derived therapeutics are currently commercially available, hiPSC-based therapeutics have yet to complete the regulatory approval process. Based on a review of the current commercially available hMSC-derived therapeutic products and upcoming hiPSC-derived products in phase 2 and 3, this paper compares the cell therapy manufacturing process between these two cell types. Moreover, the similarities as well as differences are highlighted and the resulting impact on the production process discussed. Here, emphasis is placed on (i) hMSC and hiPSC characteristics, safety, and ethical aspects, (ii) their morphology and process requirements, as well as (iii) their 2- and 3-dimensional cultivations in dependence of the applied culture medium and process mode. In doing so, also downstream processing aspects are covered and the role of single-use technology is discussed. KEY POINTS: • Mesenchymal and induced pluripotent stem cells exhibit distinct behaviors during cultivation • Single-use stirred bioreactor systems are preferred for the cultivation of both cell types • Future research should adapt and modify downstream processes to available single-use devices.

Computational fluid dynamics (CFD) is a widely used tool for investigating fluid flows in bioreactors. It has been used in the biopharmaceutical industry for years and has established itself as an important tool for process engineering characterisation. As a result, CFD simulations are increasingly being used to complement classical process engineering investigations in the laboratory with spatially and temporally resolved results, or even replace them when laboratory investigations are not possible. Parameters that can be determined include the specific power input, Kolmogorov length, hydrodynamic stress, mixing time, oxygen transfer rate, and for cultivations with microcarriers, the NS1 criterion. In the first part of this series, a literature review illustrates how these parameters can be determined using CFD and how they can be validated experimentally. In addition, an overview of the hardware and software typically used for bioreactor characterisation will also be provided, including process engineering parameter investigations from the literature. In the second part of this series, the authors’ research results will be used to show how the process engineering characterisation of mechanically driven bioreactors for the biopharmaceutical industry (stirred, orbitally shaken, and wave-mixed) can be determined and validated using CFD.

Scaling bioprocesses remains a major challenge. Since it is physically impossible to increase all process parameters equally, a suitable scale-up strategy must be selected for a successful bioprocess. One of the most widely used criteria when scaling up bioprocesses is the specific power input. However, this represents only an average value. This study aims to determine the Kolmogorov length scale distribution by means of computational fluid dynamics (CFD) and to use it as an alternative scale-up criterion for geometrically non-similar bioreactors for the first time. In order to obtain a comparable Kolmogorov length scale distribution, an automated geometry and process parameter optimization was carried out using the open-source tools OpenFOAM and DAKOTA. The Kolmogorov–Smirnov test statistic was used for optimization. A HEK293-F cell expansion (batch mode) from benchtop (Infors Minifors 2 with 4 L working volume) to pilot scale (D-DCU from Sartorius with 30 L working volume) was carried out. As a reference cultivation, the classical scale-up approach with constant specific power input (233 W m−3) was used, where a maximum viable cell density (VCDmax) of 5.02·106 cells mL−1 was achieved (VCDmax at laboratory scale 5.77·106 cells mL−1). Through the automated optimization of the stirrer geometry (three parameters), position and speed, comparable cultivation results were achieved as in the small scale with a maximum VCD of 5.60·106 cells mL−1. In addition, even on the pilot scale, cell aggregate size distribution was seen to strictly follow a geometric distribution and can be predicted with the help of CFD with the previously published correlation.

A group of the DECHEMA working group "Single-Use Technology in Biopharmaceutical Manufacture" is investigating the potential use of single-use systems for cellular agricultural applications. The preliminary results are summarized in this article.

Due to the steadily rising number of students and the simultaneous reduction in practical lessons in study programs, the need for alternative learning resources is increasing. In this work, a method is described which allows real laboratory equipment to be represented as web-based models, using a stirred bioreactor as an example. The bioreactor model that was developed allows students to explore the design and operation of stirred bioreactors independently of time, location and end device. Information associated with each bioreactor component is displayed on the model to help students become familiar with different bioreactor setups. Computational fluid dynamics (CFD) simulations were embedded to enable students to understand the influence of different stirrers on flow behavior in the bioreactors. This online tool prepares students for subsequent practical work with bioreactors in the laboratory.

Various approaches to process intensification are currently being investigated to ensure time and cost savings when producing biopharmaceuticals. In the present study, a one-step inoculum production was established based on an ultra-high cell density working cell bank with immunoglobulin G producing Chinese hamster ovary cells. Cryovials were used for direct inoculation of 1-L wave-mixed perfusion bioreactors. When reaching around 180 center dot 10(6) cells mL(-1) in N-1 perfusion mode, low-seed and high-seed fed-batch experiments in shake flasks were inoculated, as was the case for 250-mL stirred single-use bioreactors. Additionally, proof-of-concept runs at 50-L and 200-L scale were successfully performed. The intensification approach presented allows manufacturing capacity to be increased by up to 50 %.

Processes involving mammalian cell cultures - especially CHO suspension cells - dominate biopharmaceutical manufacturing. These processes are usually developed in small scale orbitally shaken cultivation systems, and thoroughly characterizing these cultivation systems is crucial to their application in research and the subsequent scale-up to production processes. With the knowledge of process engineering parameters such as oxygen transfer rate, mixing time, and power input, in combination with the demands set by the biological production system, biomass growth and product yields can be anticipated and even increased. However, the available data sources for orbitally shaken cultivation systems are often incomplete and thus not sufficient enough to generate suitable cultivation requirements. Furthermore, process engineering knowledge is inapplicable if it is not linked to the physiological demands of the cells. In the current study, a simple yet comprehensive approach for the characterization and design space prediction of orbitally shaken single-use cultivation systems is presented, including the "classical" Erlenmeyer shake flask, the cylindrical TubeSpin bioreactor and the alternately designed Optimum Growth flask. Cultivations were performed inside and outside the design space to validate the defined culture conditions, so that cultivation success (desired specific growth rates and viable cell densities) could be achieved for each cultivation system.