Shear stress analysis of mammalian cell suspensions for prediction of industrial centrifugation and its verification

This article describes the use of ultra scale-down studies requiring milliliter quantities of process material to study the clarification of mammalian cell culture broths using industrial-scale continuous centrifuges during the manufacture of a monoclonal antibody for therapeutic use. Samples were pretreated in a small high-speed rotating-disc device in order to mimic the effect on the cells of shear stresses in the feed zone of the industrial scale centrifuges. The use of this feed mimic was shown to predict a reduction of the clarification efficiency by significantly reducing the particle size distribution of the mammalian cells. The combined use of the rotating-disc device and a laboratory-scale test tube centrifuge successfully predicted the separation characteristics of industrial-scale, disc stack centrifuges operating with different feed zones. A 70% reduction in flow rate in the industrial-scale centrifuge was shown to arise from shear effects. A predicted 2.5-fold increase in throughput for the same clarification performance, achieved by the change to a centrifuge using a feed zone designed to give gentler acceleration of the bioprocess fluid, was also verified at large-scale.     

Streamlining process characterization efforts using the high throughput ambr® crossflow system for ultrafiltration and diafiltration processing of monoclonal antibodies

Commercial process development for biopharmaceuticals often involves process characterization (PC) studies to gain process knowledge and understanding in preparation for process validation. One common approach to conduct PC activities is by using design-of-experiment, which can help determine the impact process parameter deviations may have on product quality attributes. Qualified scale-down systems are typically used to conduct these studies. For an ultrafiltration/diafiltration (UF/DF) application, however, a traditional scale-down still requires hundreds of milliliters of material per run and can only conduct one experiment at a time. This poses a challenge in resources as there could be 20+ experiments required for a typical UF/DF PC study. One solution to circumvent this is the use of high-throughput systems, which enable parallel experimentation by only using a fraction of the resources. Sartorius Stedim Biotech has recently commercialized the ambr® crossflow high-throughput system to meet this need. In this study, the performance of this system during a monoclonal antibody UF/DF step was first compared with a pilot- and a manufacturing-scale tangential flow filtration (TFF) system at a single operating condition. Due to material limitations, it was then compared to only the pilot-scale TFF system across wider ranges of transmembrane pressure; crossflow rate; and diafiltration concentration in a PC study. Permeate flux, aggregate content, process yield, pH/conductivity traces, retentate concentration, axial pressure drop, and turbidity values were measured at both scales. A good agreement was attained across scales, further supporting its potential use as a scale-down system.     

Mimic of a large‐scale diafiltration process by using ultra scale‐down rotating disc filter

Ultra scale‐down (USD) approach is a powerful tool to predict large‐scale process performance by using very small amounts of material. In this article, we present a method to mimic flux and transmission performance in a labscale crossflow operation by an USD rotating disc filter (RDF). The Pellicon 2 labscale system used for evaluation of the mimic can readily be related to small pilot and industrial scale. Adopted from the pulsed sample injection technique by Ghosh and Cui (J Membr Sci. 2000;175:5‐84), the RDF has been modified by building in inserts to allow the flexibility of the chamber volume, so that only 1.5 mL of processing material is required for each diafiltration experiment. The reported method enjoys the simplicity of dead‐end mode operation with accurate control of operation conditions that can mimic well the crossflow operation in large scale. Wall shear rate correlations have been established for both the labscale cassette and the USD device, and a mimic has been developed by operating both scales under conditions with equivalent averaged shear rates. The studies using E. coli lysate show that the flux vs. transmembrane pressure profile follows a first‐order model, and the transmission of antibody fragment (Fab′) is independent of transmembrane pressure. Predicted flux and transmission data agreed well with the experimental results of a labscale diafiltration where the cassette resistance was considered. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Prediction of shear damage of plasmid DNA in pump and centrifuge operations using an ultra scale-down device

Supercoiled circular (SC) plasmid DNA is often subjected to fluid stress in large-scale manufacturing processes. It is thus important to characterize the engineering environment within a particular unit operation as well as within the associated ancillary equipment during process design for plasmid DNA manufacture so as to avoid shear-induced degradation of the SC isoform, which would compromise product efficacy in therapeutic applications. In the past few years, ultra scale-down (USD) tools were developed within our laboratory to mimic the engineering environments experienced by biomolecules within a range of manufacturing-scale ancillary, primary recovery, and purification operations, using milliliter quantities of material. Through the use of a USD shear device, the effect of elongational strain rate on SC plasmid DNA degradation was studied in this paper, and from that, the impact of a centrifugal pump, a Mono pump, and a disk-stack centrifuge feed zone on SC plasmid DNA degradation was predicted and experimentally verified at scale. Model predictions, over the range of conditions studied, were in good agreement with experimental values, demonstrating the potential of the USD approach as a decisional tool during bioprocess design.     

Prediction and verification of centrifugal dewatering of P. pastoris fermentation cultures using an ultra scale-down approach

Recent years have seen a dramatic rise in fermentation broth cell densities and a shift to extracellular product expression in microbial cells. As a result, dewatering characteristics during cell separation is of importance, as any liquor trapped in the sediment results in loss of product, and thus a decrease in product recovery. In this study, an ultra scale-down (USD) approach was developed to enable the rapid assessment of dewatering performance of pilot-scale centrifuges with intermittent solids discharge. The results were then verified at scale for two types of pilot-scale centrifuges: a tubular bowl equipment and a disk-stack centrifuge. Initial experiments showed that employing a laboratory-scale centrifugal mimic based on using a comparable feed concentration to that of the pilot-scale centrifuge, does not successfully predict the dewatering performance at scale (P-value <0.05). However, successful prediction of dewatering levels was achieved using the USD method (P-value ≥0.05), based on using a feed concentration at small-scale that mimicked the same height of solids as that in the pilot-scale centrifuge. Initial experiments used Baker's yeast feed suspensions followed by fresh Pichia pastoris fermentation cultures. This work presents a simple and novel USD approach to predict dewatering levels in two types of pilot-scale centrifuges using small quantities of feedstock (<50 mL). It is a useful tool to determine optimal conditions under which the pilot-scale centrifuge needs to be operated, reducing the need for repeated pilot-scale runs during early stages of process development.     

Adapted ultra scale-down approach for predicting the centrifugal separation behavior of high cell density cultures

The work presented here describes an ultra scale-down (USD) methodology for predicting centrifugal clarification performance in the case of high cell density fermentation broths. Existing USD approaches generated for dilute systems led to a 5- to 10-fold overprediction of clarification performance when applied to such high cell density feeds. This is due to increased interparticle forces, leading to effects such as aggregation, flocculation, or even blanket sedimentation, occurring in the low shear environment of a laboratory centrifuge, which will not be apparent in the settling region of a continuous-flow industrial centrifuge. A USD methodology was created based upon the dilution of high solids feed material to approximately 2% wet wt/vol prior to the application of the clarification test. At this level of dilution cell-cell interactions are minimal. The dilution alters the level of hindered settling in the feed suspensions, and so mathematical corrections are applied to the resultant clarification curves to mimic the original feed accurately. The methodology was successfully verified: corrected USD curves accurately predicted pilot-scale clarification performance of high cell density broths of Saccharomyces cerevisiae and Escherichia coli cells. The USD method allows for the rapid prediction of large-scale clarification of high solids density material using millilitre quantities of feed. The advantages of this method to the biochemical engineer, such as the enabling of rapid process design and scale-up, are discussed.     

Use of focused acoustics for cell disruption to provide ultra scale-down insights of microbial homogenization and its bioprocess impact--recovery of antibody fragments from rec E. coli

An ultra scale-down (USD) device that provides insight of how industrial homogenization impacts bioprocess performance is desirable in the biopharmaceutical industry, especially at the early stage of process development where only a small quantity of material is available. In this work, we assess the effectiveness of focused acoustics as the basis of an USD cell disruption method to mimic and study high-pressure, step-wise homogenization of rec Escherichia coli cells for the recovery of an intracellular protein, antibody fragment (Fab'). The release of both Fab' and of overall protein follows first-order reaction kinetics with respect to time of exposure to focused acoustics. The rate constant is directly proportional to applied electrical power input per unit volume. For nearly total protein or Fab' release (>99%), the key physical properties of the disruptate produced by focused acoustics, such as cell debris particle size distribution and apparent viscosity show good agreement with those for homogenates produced by high-pressure homogenization operated to give the same fractional release. The only key difference is observed for partial disruption of cells where focused acoustics yields a disruptate of lower viscosity than homogenization, evidently due to a greater extent of polynucleic acids degradation. Verification of this USD approach to cell disruption by high-pressure homogenization is achieved using USD centrifugation to demonstrate the same sedimentation characteristics of disruptates prepared using both the scaled-down focused acoustic and the pilot-scale homogenization methods for the same fraction of protein release.     

Performance prediction of industrial centrifuges using scale-down models

Computational fluid dynamics was used to model the high flow forces found in the feed zone of a multichamber-bowl centrifuge and reproduce these in a small, high-speed rotating disc device. Linking the device to scale-down centrifugation, permitted good estimation of the performance of various continuous-flow centrifuges (disc stack, multichamber bowl, CARR Powerfuge) for shear-sensitive protein precipitates. Critically, the ultra scale-down centrifugation process proved to be a much more accurate predictor of production multichamber-bowl performance than was the pilot centrifuge.     

Prediction of the pilot-scale recovery of a recombinant yeast enzyme using integrated models

This article describes the rapid prediction of recovery process performance for a new recombinant enzyme product on the basis of a broad portfolio of computer models and highly targeted experimentation. A process model for the recombinant system was generated by linking unit operation models in an integrated fashion, with required parameter estimation and physical property determination accomplished using data from scale-down studies. This enabled the generic modeling framework established for processing of a natural enzyme from bakers' yeast to be applied. An experimental study of the same operations at the pilot scale showed that the process model gave a conservative prediction of recombinant enzyme recovery. The model successfully captured interactions leading to a low overall product yield and indicated the need for further study of precipitate breakage in the feed zone of a disc stack centrifuge in order to improve performance. The utility of scale-down units as an aid to fast model generation and the advantage of integrating computer modeling and scale-down studies to accelerate bioprocess development are highlighted.     

Ultra scale‐down prediction using microwell technology of the industrial scale clarification characteristics by centrifugation of mammalian cell broths

This article describes how a combination of an ultra scale‐down (USD) shear device feeding a microwell centrifugation plate may be used to provide a prediction of how mammalian cell broth will clarify at scale. In particular a method is described that is inherently adaptable to a robotic platform and may be used to predict how the flow rate and capacity (equivalent settling area) of a centrifuge and the choice of feed zone configuration may affect the solids carry over in the supernatant. This is an important consideration as the extent of solids carry over will determine the required size and lifetime of a subsequent filtration stage or the passage of fine particulates and colloidal material affecting the performance and lifetime of chromatography stages. The extent of solids removal observed in individual wells of a microwell plate during centrifugation is shown to correlate with the vertical and horizontal location of the well on the plate. Geometric adjustments to the evaluation of the equivalent settling area of individual wells (Σ_M) results in an improved prediction of solids removal as a function of centrifuge capacity. The USD centrifuge settling characteristics need to be as for a range of equivalent flow rates as may be experienced at an industrial scale for a machine of different shear characteristics in the entry feed zone. This was shown to be achievable with two microwell‐plate based measurements and the use of varying fill volumes in the microwells to allow the rapid study of a fivefold range of equivalent flow rates (i.e., at full scale for a particular industrial centrifuge) and the effect of a range of feed configurations. The microwell based USD method was used to examine the recovery of CHO‐S cells, prepared in a 5 L reactor, at different points of growth and for different levels of exposure to shear post reactor. The combination of particle size distribution measurements of the cells before and after shear and the effect of shear on the solids remaining after centrifugation rate provide insight into the state of the cells throughout the fermentation and the ease with which they and accumulated debris may be removed by continuous centrifugation. Hence bioprocess data are more readily available to help better integrate cell culture and cell removal stages and resolve key bioprocess design issues such as choice of time of harvesting and the impact on product yield and contaminant carry over. Operation at microwell scale allows data acquisition and bioprocess understanding over a wide range of operating conditions that might not normally be achieved during bioprocess development. Biotechnol. Bioeng. 2009; 104: 321–331 © 2009 Wiley Periodicals, Inc.     

An ultra scale‐down characterization of low shear stress primary recovery stages to enhance selectivity of fusion protein recovery from its molecular variants

Fusion proteins offer the prospect of new therapeutic products with multiple functions. The primary recovery is investigated of a fusion protein consisting of modified E2 protein from hepatitis C virus fused to human IgG1 Fc and expressed in a Chinese hamster ovary (CHO) cell line. Fusion protein products inevitably pose increased challenge in preparation and purification. Of particular concerns are: (i) the impact of shear stress on product integrity and (ii) the presence of product‐related contaminants which could prove challenging to remove during the high resolution purification steps. This paper addresses the use of microwell‐based ultra scale‐down (USD) methods to develop a bioprocess strategy focused on the integration of cell culture and cell removal operations and where the focus is on the use of operations which impart low shear stress levels even when applied at eventual manufacturing scale. An USD shear device was used to demonstrate that cells exposed to high process stresses such as those that occur in the feed zone of a continuous non‐hermetic centrifuge resulted in the reduction of the fusion protein and also the release of glycosylated intracellular variants. In addition, extended cell culture resulted in release of such variants. USD mimics of low shear stress, hydrohermetic feed zone centrifugation and of depth filtration were used to demonstrate little to no release during recovery of these variants with both results verified at pilot scale. Furthermore, the USD studies were used to predict removal of contaminants such as lipids, nucleic acids, and cell debris with, for example, depth filtration delivering greater removal than for centrifugation but a small (～10%) decrease in yield of the fusion protein. These USD observations of product recovery and carryover of contaminants were also confirmed at pilot scale as was also the capacity or throughput achievable for continuous centrifugation or for depth filtration. The advantages are discussed of operating a lower yield cell culture and a low shear stress recovery process in return for a considerably less challenging purification demand. Biotechnol. Bioeng. 2013; 110: 1973–1983. © 2013 Wiley Periodicals, Inc.     

Purification of a conjugated polysaccharide vaccine using tangential flow diafiltration

Conjugated vaccines prepared from the capsular polysaccharide of Streptococcus pneumoniae can provide immunization against invasive pneumococcal disease, meningitis, and otitis media. One of the critical steps in the production of these vaccines is the removal of free (unreacted) polysaccharides from the protein-polysaccharide conjugate. Experimental studies were performed to evaluate the effects of membrane pore size, filtrate flux, and solution conditions on the transmission of both the conjugate and free polysaccharide through different ultrafiltration membranes. Conjugate purification was done using diafiltration performed in a linearly-scalable tangential flow filtration cassette. More than 98% of the free polysaccharide was removed within a 5-diavolume diafiltration process, which is a significant improvement over previously reported results for purification of similar conjugated vaccines. These results clearly demonstrate the opportunities for using ultrafiltration/diafiltration for the final purification of conjugated vaccine products.     

Ultra scale-down approach for the prediction of full-scale recovery of ovine polycolonal immunoglobulins used in the manufacture of snake venom-specific Fab fragment

In this article, we describe a new approach that allows the prediction of the performance of a large-scale integrated process for the primary recovery of a therapeutic antibody from an analysis of the individual unit operations and their interactions in an ultra scale-down mimic of the process. The recovery process consisted of four distinct unit operations. Using the new approach we defined the important engineering parameters in each operation that impacted the overall recovery process and in each case verified its effect by a combination of modelling and experimentation. Immunoglobulins were precipitated from large volumes of dilute blood plasma and the precipitated flocs were recovered by centrifugal separation from the liquor containing contaminating proteins, including albumin. The fluid mechanical forces acting on the precipitate and the time of exposure to these forces were used to define a time-integrated fluid stress. This was used as a scaling factor to predict the properties of the precipitated flocs at large scale. In the case of centrifugation, the performance of a full-scale disc stack centrifuge was predicted. This was achieved from a computational fluid dynamics (CFD) analysis of the flow field in the centrifuge coupled with experimental data obtained from the precipitated immunoglobulin flocs using the scale-down precipitation tank, a rotating shear device, and a standard swing-out rotor centrifuge operating under defined conditions. In this way, the performance of the individual unit operations, and their linkage, was successfully analysed from a combination of modelling and experiments. These experiments required only millilitre quantities of the process material. The overall performance of the large-scale process was predicted by tracking the changes in physical and biological properties of the key components in the system, including the size distribution of the antibody precipitates and antibody activity through the individual unit operations in the ultra scale-down process flowsheet.     

Characterization and feasibility of a miniaturized stirred tank bioreactor to perform E. coli high cell density fed-batch fermentations

The use of small scale bioreactors that are mechanically and functionally similar to large scale reactors is highly desirable to accelerate bioprocess development because they enable well-defined scale translations. In this study, a 25-mL miniaturized stirred tank bioreactor (MSBR) has been characterized in terms of its power input, hydrodynamics, and volumetric oxygen transfer coefficient (k(L)a) to assess its potential to grow high cell density (HCD) cultures using adequate scale-down criteria. Engineering characterization results show scale down, based on matched specific power input (P(G)/V), is feasible from a 20-L pilot scale stirred tank bioreactor. Results from fed-batch fermentations performed using Fab' producing E. coli W3110 at matched (P(G)/V) in the MSBR and 20-L STR demonstrated that the MSBR can accurately scale down the 20-L fermentation performance in terms of growth and Fab' production. Successful implementation of a fed-batch strategy in the MSBR resulted in maximum optical density of ca. 114 and total Fab' concentration of 940 μg/mL compared with ca. 118 and 990 μg/mL in 20-L STR. Furthermore, the use of the MSBR in conjunction with primary recovery scale-down tools to assess the harvest material of both reactors showed comparable shear sensitivity and centrifugation performance. The conjoint use of the MSBR with ultra scale-down (USD) centrifugation mimics can provide a cost-efficient manner in which to design and develop bioprocesses that account for good upstream performance as well as their manufacturability downstream.     

Purification of hemoglobin by tangential flow filtration with diafiltration

A recent study by Palmer, Sun, and Harris (Biotechnol. Prog., 25:189–199, 2009) demonstrated that tangential flow filtration (TFF) can be used to produce HPLC‐grade bovine and human hemoglobin (Hb). In this current study, we assessed the quality of bovine Hb (bHb) purified by introducing a 10 L batch‐mode diafiltration step to the previously mentioned TFF Hb purification process. The bHb was purified from bovine red blood cells (RBCs) by filtering clarified RBC lysate through 50 nm (stage I) and 500 kDa (stage II) hollow fiber (HF) membranes. The filtrate was then passed through a 100 kDa (stage III) HF membrane with or without an additional 10 L diafiltration step to potentially remove additional small molecular weight impurities. Protein assays, SDS‐PAGE, and LC‐MS of the purified bHb (stage III retentate) reveal that addition of a diafiltration step has no effect on bHb purity or yield; however, it does increase the methemoglobin level and oxygen affinity of purified bHb. Therefore, we conclude that no additional benefit is gained from diafiltration at stage III and a three stage TFF process is sufficient to produce HPLC‐grade bHb. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

A methodical approach to ultra-scale-down of process sequences: application to casein removal from the milk of transgenic animals

Scale-down models of individual operations are widely used in biopharmaceutical process development to obtain information about the performance of production-scale equipment on the basis of inexpensive and efficient laboratory-scale tests, for the purposes of validation or optimization or characterization studies. We have investigated the ability of scale-down models of whole process sequences to provide reliable information for process scale-up from laboratory- to pilot-scales of operation. Using the example of the recovery of a protein from transgenic milk, we have conducted an a priori scale-down analysis of a projected pilot-scale process sequence. A systematic approach was developed to ensure that all critical aspects of process behavior were included in the scale-down model, resulting in the creation of an accurate and reliable scale-down representation of the pilot-scale process. The data from scale-down process trials conducted at 70 and 200 mL scales of operation served to highlight crucial factors determining process performance, and proved reliable in predicting the performance of the pilot-scale process over a scaling factor of 1000.     

Removal of RNA impurities by tangential flow filtration in an RNase-free plasmid DNA purification process

Addition of animal-derived ribonuclease A to degrade RNA impurities is not recommended in the manufacture of pharmaceutical-grade plasmid DNA. Tangential flow filtration (TFF) takes advantage of the significant size difference between RNA and plasmid DNA to remove RNA in the permeate while plasmid remains in the retentate, in an RNase-free plasmid purification process. Operating conditions including transmembrane pressure, membrane pore size, conductivity of the diafiltration buffer, and plasmid load on the membrane were investigated to maximize RNA clearance. Although direct TFF of clarified lysate removed substantial amounts of RNA, the RNA levels left in the retentate were still significant. Calcium chloride is a potent precipitant of high-molecular-weight RNA. The addition of calcium chloride to the clarified lysate combined with the clearance of low-molecular-weight RNA by TFF resulted in complete RNA removal and high plasmid recovery.     

A model-based framework for parallel scale-down fed-batch cultivations in mini-bioreactors for accelerated phenotyping

Concentration gradients that occur in large industrial-scale bioreactors due to mass transfer limitations have significant effects on process efficiency. Hence, it is desirable to investigate the response of strains to such heterogeneities to reduce the risk of failure during process scale-up. Although there are various scale-down techniques to study these effects, scale-down strategies are rarely applied in the early developmental phases of a bioprocess, as they have not yet been implemented on small-scale parallel cultivation devices. In this study, we combine mechanistic growth models with a parallel mini-bioreactor system to create a high-throughput platform for studying the response of Escherichia coli strains to concentration gradients. As a scaled-down approach, a model-based glucose pulse feeding scheme is applied and compared with a continuous feed profile to study the influence of glucose and dissolved oxygen gradients on both cell physiology and incorporation of noncanonical amino acids into recombinant proinsulin. The results show a significant increase in the incorporation of the noncanonical amino acid norvaline in the soluble intracellular extract and in the recombinant product in cultures with glucose/oxygen oscillations. Interestingly, the amount of norvaline depends on the pulse frequency and is negligible with continuous feeding, confirming observations from large-scale cultivations. Most importantly, the results also show that a larger number of the model parameters are significantly affected by the scale-down scheme, compared with the reference cultivations. In this example, it was possible to describe the effects of oscillations in a single parallel experiment. The platform offers the opportunity to combine strain screening with scale-down studies to select the most robust strains for bioprocess scale-up.     

A new microfluidic concept for parallel operated milliliter-scale stirred tank bioreactors

Parallel miniaturized stirred tank bioreactors are an efficient tool for "high-throughput bioprocess design." As most industrial bioprocesses are pH-controlled and/or are operated in a fed-batch mode, an exact scale-down of these reactions with continuous dosing of fluids into the miniaturized bioreactors is highly desirable. Here, we present the development, characterization, and application of a novel concept for a highly integrated microfluidic device for a bioreaction block with 48 parallel milliliter-scale stirred tank reactors (V = 12 mL). The device consists of an autoclavable fluidic section to dispense up to three liquids individually per reactor. The fluidic section contains 144 membrane pumps, which are magnetically driven by a clamped-on actuator section. The micropumps are designed to dose 1.6 μL per pump lift. Each micropump enables a continuous addition of liquid with a flow rate of up to 3 mL h(-1) . Viscous liquids up to a viscosity of 8.2 mPa s (corresponds to a 60% v/v glycerine solution) can be pumped without changes in the flow rates. Thus, nearly all feeding solutions can be delivered, which are commonly used in bioprocesses. The functionality of the first prototype of this microfluidic device was demonstrated by double-sided pH-controlled cultivations of Saccharomyces cerevisiae based on signals of fluorimetric sensors embedded at the bottom of the bioreactors. Furthermore, fed-batch cultivations with constant and exponential feeding profiles were successfully performed. Thus, the presented novel microfluidic device will be a useful tool for parallel and, thus, efficient optimization of controlled fed-batch bioprocesses in small-scale stirred tank bioreactors. This can help to reduce bioprocess development times drastically.     

Computational fluid dynamics simulation improves the design and characterization of a plug-flow-type scale-down reactor for microbial cultivation processes

The scale-up of bioprocesses remains one of the major obstacles in the biotechnology industry. Scale-down bioreactors have been identified as valuable tools to investigate the heterogeneities observed in large-scale tanks at the laboratory scale. Additionally, computational fluid dynamics (CFD) simulations can be used to gain information about fluid flow in tanks used for production. Here, we present the rational design and comprehensive characterization of a scale-down setup, in which a flexible and modular plug-flow reactor was connected to a stirred-tank bioreactor. With the help of CFD using the realizable k-ε model, the mixing time difference between a 20 and 4000 L bioreactor was evaluated and used as scale-down criterion. CFD simulations using a shear stress transport (SST) k-ω turbulence model were used to characterize the plug-flow reactor in more detail, and the model was verified using experiments. Additionally, the model was used to simulate conditions where experiments technically could not be performed due to sensor limitations. Nevertheless, verification is difficult in this case as well. This was the first time a scale-down setup was tested on high-cell-density Escherichia coli cultivations to produce industrially relevant antigen-binding fragments (Fab). Biomass yield was reduced by 11% and specific product yield was reduced by 20% during the scale-down cultivations. Additionally, the intracellular Fab fraction was increased by using the setup. The flexibility of the introduced scale-down setup in combination with CFD simulations makes it a valuable tool for investigating scale effects at the laboratory scale. More information about the large scale is still necessary to further refine the setup and to speed up bioprocess scale-up in the future.