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Single-use systems have become increasingly important in recent years. This applies in particular to their use in the manufacture of biopharmaceutical products based on mammalian cell cultures, such as antibodies for the therapy of cancer and autoimmune diseases. Today, developers and manufacturers of such biotherapeutics can choose from a large product portfolio of single-use systems from different suppliers. For example, there are single-use versions of storage bags, filters, mixers, bioreactors, connectors, harvesting and transfer systems, membrane adsorbers, freeze- and -thaw systems, to name just a few examples. Thus, the production of clinical samples in complete single-use production facilities (upstream processing to filling) is already a reality. Based on the definition of the term and the presentation of milestones in the development of single-use technology, the book chapter discusses the most important advantages and existing limitations of the single-use systems currently available on the market. Subsequently, the current state of development of single-use technology is presented on the basis of selected product examples. The focus is on the upstream area and single-use bioreactors. Finally, approaches for process intensification up to complete continuous production resulting from the increasing implementation of single-use systems are deduced.

In recent years, the use of hMSCs, which may be isolated from adipose tissue among others, for the treatment of diseases has increased significantly. The cell quantities required for such therapeutic approaches, between 1012 and 1013, have thus far been predominantly produced using commercially available multi-tray systems, such as the Cell Factory (Thermo Fisher Scientific) or HYPERStack (Corning), which can be purchased with up to 40 layers. However, the handling of these planar multilayer systems is difficult, and process monitoring opportunities remain limited. Here, automated stirred single-use bioreactors provide a viable alternative to the time-consuming multiplication of cells using such planar systems, while still managing to achieve the desired clinically relevant quantities. In these stirred single-use systems, adherent cells are predominantly cultivated in suspension up to pilot scale using carrier materials, also referred to as microcarriers (MCs).This chapter describes the steps which need to be realized to guarantee successful hMSC expansion within a stirred single-use bioreactor (Eppendorf's BioBLU® 0.3c) operated using MCs under serum- and xeno-free conditions at benchtop scale. The cultivations were performed using an immortalized human adipose-derived mesenchymal stem cell (hASC) line, hence referred to as hASC52telo, and a new chemically defined, xeno-free medium, hence referred to as the UrSuppe formulation. Spinner flask cultivations were performed under comparable process conditions.

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during rotation. However, the experimental determination of the power input in small-scale vessels is still challenging due to relatively high friction losses inside typically used bushings, bearings and/or shaft seals and the accuracy of commercially available torque meters. Thus, only limited data for small-scale bioreactors, in particular single-use systems, is available in the literature, making comparisons among different single-use systems and their conventional counterparts difficult. This manuscript provides a protocol on how to measure power inputs in benchtop scale bioreactors over a wide range of turbulence conditions, which can be described by the dimensionless Reynolds number (Re). The aforementioned friction losses are effectively reduced by the use of an air bearing. The procedure on how to set up, conduct and evaluate a torque-based power input measurement, with special focus on cell culture typical agitation conditions with low to moderate turbulence (100 < Re < 2·104), is described in detail. The power input of several multi-use and single-use bioreactors is provided by the dimensionless power number (also called Newton number, P0), which is determined to be in the range of P0 ≈ 0.3 and P0 ≈ 4.5 for the maximum Reynolds numbers in the different bioreactors.

Cellular agriculture is the controlled and sustainable manufacture of agricultural products with cells and tissues without plant or animal involvement. Today, microorganisms cultivated in bioreactors already produce egg and milk proteins, sweeteners, and flavors for human nutrition as well as leather and fibers for shoes, bags, and textiles. Furthermore, plant cell and tissue cultures provide ingredients that stimulate the immune system and improve skin texture, with another precommercial cellular agriculture product, in vitro meat, currently receiving a great deal of attention. All these approaches could assist traditional agriculture in continuing to provide for the dietary requirements of a growing world population while freeing up important resources such as arable land. Despite early successes, challenges remain and are discussed in this review, with a focus on production processes involving plant and animal cell and tissue cultures.

Human adipose stem cells (hASCs) are promising candidates for cell-based therapies, but they need to be efficiently expanded in vitro as they cannot be harvested in sufficient quantities. Recently, dynamic bioreactor systems operated with microcarriers achieved considerable high cell densities. Thus, they are a viable alternative to static planar cultivation systems to obtain high numbers of clinical-grade hASCs. Nevertheless, the production of considerable biomass in a short time must not be achieved to the detriment of the cells’ quality. To facilitate the scalable expansion of hASC, we have developed a new serum- and xeno-free medium (UrSuppe) and a biodegradable microcarrier (BR44). In this study, we investigated whether the culture of hASCs in defined serum-free conditions on microcarriers (3D) or on planar (2D) cell culture vessels may influence the expression of some marker genes linked with the immature degree or the differentiated status of the cells. Furthermore, we investigated whether the biomaterials, which form our biodegradable MCs, may affect cell behavior and differentiation. The results confirmed that the quality and the undifferentiated status of the hASCs are very well preserved when they grow on BR44 MCs in defined serum-free conditions. Indeed, the ASCs showed a gene expression profile more compatible with an undifferentiated status than the same cells grown under standard planar conditions.

This chapter aims to give an overview of instrumented single‐use bioreactor (SUB) types that are currently available commercially. Based on the milestones in the development of SUBs, it focuses on the three most often applied types: wave‐mixed, stirred, and orbitally shaken versions. Their typical working principles are explained, possible fields of application are deduced, and trends in their selection are summarized. The development of SUBs can be described in three phases: the early beginnings; the establishment of disposable membrane bioreactors, multitray cell culture systems, and the first bag bioreactors; and the expansion of wave‐mixed, stirred, orbitally shaken, and further SUBs. The most suitable bioreactor for a desired application depends on several factors. The selection of the bioreactor is significantly influenced by the cultivation task, the engineering parameters of the bioreactor, the scale, the production organism, the legal requirements, the infrastructure, the know‐how of staff, and the costs for investment and operation.

Adipose tissue is an abundant source of stem cells. However, liposuction cannot yield cell quantities sufficient for direct applications in regenerative medicine. Therefore, the development of GMP-compliant ex vivo expansion protocols is required to ensure the production of a “cell drug” that is safe, reproducible, and cost-effective. Thus, we developed our own basal defined xeno- and serum-free cell culture medium (UrSuppe), specifically formulated to grow human adipose stem cells (hASCs). With this medium, we can directly culture the stromal vascular fraction (SVF) cells in defined cell culture conditions to obtain hASCs. Cells proliferate while remaining undifferentiated, as shown by Flow Cytometry (FACS), Quantitative Reverse Transcription PCR (RT-qPCR) assays, and their secretion products. Using the UrSuppe cell culture medium, maximum cell densities between 0.51 and 0.80 × 105 cells/cm2 (=2.55–4.00 × 105 cells/mL) were obtained. As the expansion of hASCs represents only the first step in a cell therapeutic protocol or further basic research studies, we formulated two chemically defined media to differentiate the expanded hASCs in white or beige/brown adipocytes. These new media could help translate research projects into the clinical application of hASCs and study ex vivo the biology in healthy and dysfunctional states of adipocytes and their precursors. Following the cell culture system developers’ practice and obvious reasons related to the formulas’ patentability, the defined media’s composition will not be disclosed in this study.

Stirred single-use bioreactors in combination with microcarriers (MCs) have established themselves as a technology that has the potential to meet the demands of current and future cell therapeutic markets. However, most of the published processes have been performed using fetal bovine serum (FBS) containing cell culture medium and non-biocompatible MCs. This approach has two significant drawbacks: firstly, the inevitable potential risks associated with the use of FBS for clinical applications; secondly, non-biocompatible MCs have to be removed from the cell suspension before implantation, requiring a step that causes loss of viable cells and adds further costs and complications. This study aimed to develop a new platform based on a chemically defined xeno- and serum-free cell culture medium and biodegradable MC that can support the growth of human adipose stem cells (hASCs) while still preserving their undifferentiated status. A specific combination of components and manufacturing parameters resulted in a MC prototype, called “BR44”, which delivered the desired functionality. MC BR44 allows the hASCs to stick to its surface and grow in a chemically defined xeno- and serum-free medium (UrSuppe). Although the cells’ expansion rate was not as high as with a commercial non-biodegradable standard MC, those cultured on BR44 maintained a better undifferentiated status in both static and dynamic conditions than those cultured on traditional 2D surfaces.

Human adipose tissue-derived stromal/stem cells (hASC) represent a valuable source of cells for clinical applications, especially in the field of regenerative medicine. Therefore, it comes as no surprise that interest in hASCs has increased greatly over the last decade. However, in order to use hASCs successfully in clinical applications, in vitro expansion is required. Single-use bioreactors in combination with microcarriers (MC) have been shown to be suitable systems for this task (1-3). However, hASCs are prone to higher shear sensitivity than conventional cell lines (e.g. CHO, BHK) that are normally expanded in these systems. Hence, the goal of this study was to investigate the influence of different shear stress levels on the growth of hASCs in small scale single-use spinner flasks. For this purpose, Computational Fluid Dynamics simulations based on a Euler-Euler and Euler-Lagrange approach were performed to predict the hydrodynamic stresses (0.06 – 0.87 Pa), the residence times (0.4 – 7.3 s) and the circulation times (1.6 - 16.6 s) of the MCs in various high shear zones. The numerical findings were combined with experimental data from cultivation studies (0.29 – 1.1∙106 hASC/mL) in order to develop a segregated mathematical growth model for the prediction of MC-associated hASC growth in small scale single-use spinner flasks.

Human adipose stem cells (hASCs) are candidates with great potential for cell-based therapies. However, these cells need to be efficiently expanded in vitro, as they cannot be harvested in sufficient quantities from the human body. Stirred single-use bioreactors in combination with microcarriers have established themselves as a technology with potential to meet the demands of current and future cell therapeutic markets. Recent studies have shown that hASCs in stirred single-use bioreactors can be expanded up to benchtop and pilot scale and provide sufficiently high cell numbers (up to 1.2∙106 hASCs/mL) for current autologous and allogeneic therapies. However, most of the studies that provide clinically relevant cell numbers were performed with serum containing cell culture medium (5-20 % FBS). Due to the adverse effects of FBS for clinical applications, the present study focused on the development of a serum and xeno-free, chemically defined cell culture medium (called UrSuppe) for hASCs. In addition, initial efforts have been made to develop a biodegradable MC suitable for the dynamic cultivation of hASCs with UrSuppe cell culture medium in stirred single-use bioreactors. Results of cell cultivation studies with hTERT-ASCs under serum-reduced (5 % FBS) and serum-free conditions are presented and discussed for planar and MC-based (up to 3.0∙105 cells/cm2) cultivations and compared with those obtained from a kinetic growth model recently described in the scientific literature (Jossen et al. 2018). In addition, initial results of cultivations in spinner flasks with the biodegradable MC are compared with those from a commercially available polystyrene MC. In addition to characterising and comparing cell growth behaviour, special emphasis is placed on critical cell quality attributes (CQAs).

Cell-based therapies have become increasingly important in the field of regenerative medicine, as global revenues of approximately 1 billion US$ indicate. A special focus in the clinical field is placed on human mesenchymal stem cells (hMSCs), especially on those isolated from the stromal vascular fraction of the adipose tissue. Due to their good clinical efficacy and tolerability, they can be used for allogeneic cell therapies (e.g. Crohn’s disease, graft versus host disease, acute myocardial infarct). However, the required number of therapeutically active human adipose tissue-derived stromal/stem cells (hASCs) for allogeneic applications is in the range of trillion cells per year. Thus, alternatives to the 2-dimensional planar cultivation systems, typically applied to propagate hASCs are urgently required. Different studies have recently demonstrated the applicability of stirred single-use bioreactors as a promising alternative to the planar cultivation systems. In these dynamically mixed culture systems, the required growth surface for the adherent growing cells is provided by microcarriers (MCs), which are suspended in the bioreactor. Although adherent mammalian cells are well investigated in combination with MCs for the production of vaccines, working with hASCs is more complex due to their higher shear sensitivity. This raises the question of how the induced fluid shear stress affects the cell expansion, the cell quality and the formation of MC-cell agglomerates during the cultivation in dynamic bioreactors. For this purpose, MC-based cell expansions were performed in single-use spinner flasks at different impeller speeds below (25, 43 rpm) and above (90 and 120 rpm) the specific suspension criteria NS1u (49 rpm) and NS1 (63 rpm) for polystyrene-based MCs. Cell numbers, metabolites and the formation of MC-cell agglomerates were measured during the cell expansion process. Furthermore, the expression of standard surface markers (CD14, CD20, CD34, CD45, CD73, CD9

Human Adipose Tissue Stem Cells (hASCs) are a valuable source of cells for clinical applications (e.g., treatment of acute myocardial infarction and inflammatory diseases), especially in the field of regenerative medicine. However, for autologous (patient-specific) and allogeneic (off-the-shelf) hASC-based therapies, in-vitro expansion is necessary prior to the clinical application in order to achieve the required cell numbers. Safe, reproducible and economic in-vitro expansion of hASCs for autologous therapies is more problematic because the cell material changes for each treatment. Moreover, cell material is normally isolated from non-healthy or older patients, which further complicates successful in-vitro expansion. Hence, the goal of this study was to perform cell expansion studies with hASCs isolated from two different patients/donors (i.e., different ages and health statuses) under xeno- and serum-free conditions in static, planar (2D) and dynamically mixed (3D) cultivation systems. Our primary aim was I) to compare donor variability under in-vitro conditions and II) to develop and establish an unstructured, segregated growth model as a proof-of-concept study. Maximum cell densities of between 0.49 and 0.65 × 105 hASCs/cm2 were achieved for both donors in 2D and 3D cultivation systems. Cell growth under static and dynamically mixed conditions was comparable, which demonstrated that hydrodynamic stresses (P/V = 0.63 W/m3, τnt = 4.96 × 10−3 Pa) acting at Ns1u (49 rpm for 10 g/L) did not negatively affect cell growth, even under serum-free conditions. However, donor-dependent differences in the cell size were found, which resulted in significantly different maximum cell densities for each of the two donors. In both cases, stemness was well maintained under static 2D and dynamic 3D conditions, as long as the cells were not hyperconfluent. The optimal point for cell harvesting was identified as between cell densities of 0.41 and 0.56 × 105 hASCs/cm2 (end of exponen

It is undisputed that stirred bioreactors are the most common type of bioreactors for submerged bioprocesses. Stirred bioreactors for biopharmaceuticals, which represent the strongest-growing branch of modern biotechnological products, are the focus of our chapter. They are available in working volumes from milliliters up to 70 m3 and are used for a wide range of expression systems that require aseptic conditions. Based on a brief summary of some basic rules for the construction and operation of stirred bioreactors, this chapter will review key engineering issues that affect the design of aseptic stirred bioreactors. We will also provide readers with some general information on transport phenomena in stirred bioreactors before discussing special design aspects and performance parameters for expression systems (mammalian and microbial cells) that prevail in commercial biopharmaceutical production processes. Special attention will be paid to single-use stirred bioreactors, which may have advantages over their reusable counterparts and have become widely accepted over the past years.

Human adipose tissue-derived stromal/stem cells (hASCs) are a valuable source of cells for clinical applications, especially in the field of regenerative medicine. Therefore, it comes as no surprise that the interest in hASCs has greatly increased over the last decade. However, in order to use hASCs in clinically relevant numbers, in vitro expansion is required. Single-use stirred bioreactors in combination with microcarriers (MCs) have shown themselves to be suitable systems for this task. However, hASCs tend to be less robust, and thus, more shear sensitive than conventional production cell lines for therapeutic antibodies and vaccines (e.g., Chinese Hamster Ovary cells CHO, Baby Hamster Kidney cells BHK), for which these bioreactors were originally designed. Hence, the goal of this study was to investigate the influence of different shear stress levels on the growth of humane telomerase reversed transcriptase immortalized hASCs (hTERT-ASC) and aggregate formation in stirred single-use systems at the mL scale: the 125 mL (= SP100) and the 500 mL (= SP300) disposable Corning® spinner flask. Computational fluid dynamics (CFD) simulations based on an Euler⁻Euler and Euler⁻Lagrange approach were performed to predict the hydrodynamic stresses (0.06⁻0.87 Pa), the residence times (0.4⁻7.3 s), and the circulation times (1.6⁻16.6 s) of the MCs in different shear zones for different impeller speeds and the suspension criteria (Ns1u, Ns1). The numerical findings were linked to experimental data from cultivations studies to develop, for the first time, an unstructured, segregated mathematical growth model for hTERT-ASCs. While the 125 mL spinner flask with 100 mL working volume (SP100) provided up to 1.68.10⁵ hTERT-ASC/cm² (= 0.63 × 10⁶ living hTERT-ASCs/mL, EF 56) within eight days, the peak living cell density of the 500 mL spinner flask with 300 mL working volume (SP300) was 2.46 × 10⁵ hTERT-ASC/cm² (= 0.88 × 10⁶ hTERT-ASCs/mL, EF 81) and was achieved on day eight. Optimal

We review the GMP/regulatory experience with advanced therapy medicinal products (ATMPs) approximately 10 years after EC regulation 1394/2007, aka the ATMP regulation applied. Particularly, we discuss practical issues which have arisen during this period and which pertain to quantitative aspects of specifications of such products - both allogenic and autologous - and how these perceived hurdles may help in identifying truly useful products. We further review the impact the ATMP-GMP specific guideline might have and how it reflects the experience gathered. Finally, we shed light on combinations of small molecule drugs and ATMPs; we provide an example of the efficacy of such combination products as well as an outlook concerning their classifications.

Einweg‐Bioreaktoren sind in der Entwicklung und Produktion von monoklonalen Antikörpern angekommen. Studien belegen, dass sie richtig ausgewählt und eingesetzt sicherere, schnellere, grünere und kleinere Produktionsverfahren als ihre wiederverwendbaren Gegenspieler erlauben und zu Kosteneinsparungen beitragen. In der Inokulumproduktion haben sich wellendurchmischte Einweg‐Bioreaktoren durchgesetzt, während die Anwender in der Fermentation mehrheitlich mit gerührten Einweg‐Bioreaktoren arbeiten. Es wird prognostiziert, dass die Anzahl der Einweg‐Bioreaktoren weiter zunehmen wird.

Power input is an important engineering and scale-up/down criterion in stirred bioreactors. However, reliably measuring power input in laboratory-scale systems is still challenging. Even though torque measurements have proven to be suitable in pilot scale systems, sensor accuracy, resolution, and errors from relatively high levels of friction inside bearings can become limiting factors at smaller scales. An experimental setup for power input measurements was developed in this study by focusing on stainless steel and single-use bioreactors in the single-digit volume range. The friction losses inside the air bearings were effectively reduced to less than 0.5% of the measurement range of the torque meter. A comparison of dimensionless power numbers determined for a reference Rushton turbine stirrer (NP = 4.17 ± 0.14 for fully turbulent conditions) revealed good agreement with literature data. Hence, the power numbers of several reusable and single-use bioreactors could be determined over a wide range of Reynolds numbers between 100 and >104. Power numbers of between 0.3 and 4.5 (for Re = 104) were determined for the different systems. The rigid plastic vessels showed similar power characteristics to their reusable counterparts. Thus, it was demonstrated that the torque-based technique can be used to reliably measure power input in stirred reusable and single-use bioreactors at the laboratory scale.

Oxygen supply in aerobic bioprocesses is of crucial importance. For this reason, this paper presents the oxygen demand of different cells and summarizes experimental and numerical possibilities for the determination of oxygen transfer in bioreactors. The focus lies on the volumetric oxygen mass transfer coefficient (kLa) calculation using computational fluid dynamics and state-of-the-art models for surface-aerated and forced-aerated bioreactors. In addition, experimental methods for the determination of the kLa value and the gas bubble size distribution are presented.