A systematic approach for scale-down model development and characterization of commercial cell culture processes

The objective of process characterization is to demonstrate robustness of manufacturing processes by understanding the relationship between key operating parameters and final performance. Technical information from the characterization study is important for subsequent process validation, and this has become a regulatory expectation in recent years. Since performing the study at the manufacturing scale is not practically feasible, development of scale-down models that represent the performance of the commercial process is essential to achieve reliable process characterization. In this study, we describe a systematic approach to develop a bioreactor scale-down model and to characterize a cell culture process for recombinant protein production in CHO cells. First, a scale-down model using 2-L bioreactors was developed on the basis of the 2000-L commercial scale process. Profiles of cell growth, productivity, product quality, culture environments (pH, DO, pCO2), and level of metabolites (glucose, glutamine, lactate, ammonia) were compared between the two scales to qualify the scale-down model. The key operating parameters were then characterized in single-parameter ranging studies and an interaction study using this scale-down model. Appropriate operation ranges and acceptance criteria for certain key parameters were determined to ensure the success of process validation and the process performance consistency. The process worst-case condition was also identified through the interaction study.     

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.     

An ultra scale-down method to investigate monoclonal antibody processing during tangential flow filtration using ultrafiltration membranes

The availability of material for experimental studies is a key constraint in the development of full-scale bioprocesses. This is especially true for the later stages in a bioprocess sequence such as purification and formulation, where the product is at a relatively high concentration and traditional scale-down models can require significant volumes. Using a combination of critical flow regime analysis, bioprocess modelling, and experimentation, ultra scale-down (USD) methods can yield bioprocess information using only millilitre quantities before embarking on highly demanding full-scale studies. In this study the performance of a pilot-scale tangential flow filtration (TFF) system based on a membrane flat-sheet cassette using pumped flow was predicted by devising an USD device comprising a stirred cell using a rotating disc. The USD device operates with just 2.1 cm² of membrane area and, for example, just 1.7 mL of feed for diafiltration studies. The novel features of the design involve optimisation of the disc location and the membrane configuration to yield an approximately uniform shear rate. This is characterised using computational fluid dynamics for a defined layer above the membrane surface. A pilot-scale TFF device operating at ~500-fold larger feed volume and membrane area was characterised in terms of the shear rate derived from flow rate-pressure drop relationships for the cassette. Good agreement was achieved between the USD and TFF devices for the flux and resistance values at equivalent average shear rates for a monoclonal antibody diafiltration stage.     

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.     

A methodology for the design of scale-down bioreactors by the use of mixing and circulation stochastic models

Scale-up is traduced in practice by an increase of the dimensions of the bioreactors, leading to a modification of the time scale and thus of the process dynamics. In the present work, a methodology to study the effect of scale-up on bioreactors hydrodynamics and to put in place scale-down reactors representative of the flow properties encountered in real scales bioreactors is detailed.In order to simplify the analysis, we have proposed the use of a stochastic model which is directly affected by the time scale. Indeed, to run simulations with such models, we have to specify the time taken to achieve a transition Δt. Stochastic models are thus reliable to study scale-up effect on stirred reactors hydrodynamics. In addition, these models permit to have an insight on the internal dynamic of the process.In the case of the circulation process, qualitative aspects have to be taken into account and induce a modification of the flow regions arrangement of the model. The stochastic analysis of large-scale bioreactors permits to propose a translating methodology into a scale-down context. Optimised scale-down reactors can be used further to carry out fermentation tests with the hydrodynamic conditions of the industrial scale. In a general rule, the performances of stochastic model allow to facilitate greatly the analysis of the scale-up effect and the hydrodynamic characteristics of both large-scale and scale-down reactors.     

Development and qualification of a representative scale-down model of automated Ficoll-based processing of a cell-based therapeutic according to quality by design principles

Background aimsThis article describes the development of a small-scale model for Ficoll-based cell separation as part of process development of an advanced therapy medicinal product and its qualification. Because of the complexity of biological products, their manufacturing process as well as characterization and control needs to be accurately understood. Likewise, scale-down models serve as an indispensable tool for process development, characterization, optimization and validation. This scale-down model represents a cell processor device widely used in advance therapies. This approach is inteded to optimise resources and to focus its use on process characterisation studies under the paradigm of the Quality by design. A scale-down model should reflect the large manufacturing scale. Consequently, this simplified system should offer a high degree of control over the process parameters to depict a robust model, even considering the process limitations. For this reason, a model should be developed and qualified for the intended purpose.MethodsProcess operating parameters were studied, and their resulting performance at full scale was used as a baseline to guide scale-down model development. Once the model was established, comparability runs were performed by establishing standard operating conditions with bone marrow samples. These analyses showed consistency between the bench and the large scale. Additionally, statistical analyses were employed to demonstrate equivalence.ResultsThe process performance indicators and assessed quality attributes were equivalent and fell into the acceptance ranges defined for the large-scale process.ConclusionsThis scale-down model is suitable for use in process characterization studies.     

Scale-down of continuous filtration for rapid bioprocess design: Recovery and dewatering of protein precipitate suspensions

The early specification of bioprocesses often has to be achieved with small (tens of millilitres) quantities of process material. If extensive process discovery is to be avoided at pilot or industrial scale, it is necessary that scale-down methods be created that not only examine the conditions of process stages but also allows production of realistic output streams (i.e., streams truly representative of the large scale). These output streams can then be used in the development of subsequent purification operations. The traditional approach to predicting filtration operations is via a bench-scale pressure filter using constant pressure tests to examine the effect of pressure on the filtrate flux rate and filter cake dewatering. Interpretation of the results into cake resistance at unit applied pressure (alpha) and compressibility (n) is used to predict the pressure profile required to maintain the filtrate flux rate at a constant predetermined value. This article reports on the operation of a continuous mode laboratory filter in such a way as to prepare filter cakes and filtrate similar to what may be achieved at the industrial scale. Analysis of the filtration rate profile indicated the filter cake to have changing properties (compressibility) with time. Using the insight gained from the new scale-down methodology gave predictions of the flux profile in a pilot-scale candle filter superior to those obtained from the traditional batch filter used for laboratory development.     

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.     

Scale-down model qualification of ambr® 250 high-throughput mini-bioreactor system for two commercial-scale mAb processes

Recent advances in high-throughput (HTP) automated mini-bioreactor systems have significantly improved development timelines for early-stage biologic programs. Automated platforms such as the ambr® 250 have demonstrated the ability, using appropriate scale-down approaches, to provide reliable estimates of process performance and product quality from bench to pilot scale, but data sets comparing to large-scale commercial processes (>10,000 L) are limited. As development moves toward late stages, specifically process characterization (PC), a qualified scale-down model (SDM) of the commercial process is a regulatory requirement as part of Biologics License Application (BLA)-enabling activities. This work demonstrates the qualification of the ambr® 250 as a representative SDM for two monoclonal antibody (mAb) commercial processes at scales >10,000 L. Representative process performance and product quality associated with each mAb were achieved using appropriate scale-down approaches, and special attention was paid to pCO₂ to ensure consistent performance and product quality. Principal component analysis (PCA) and univariate equivalence testing were utilized in the qualification of the SDM, along with a statistical evaluation of process performance and product-quality attributes for comparability. The ambr® 250 can predict these two commercial-scale processes (at center-point condition) for cell-culture performance and product quality. The time savings and resource advantages to performing PC studies in a small-scale HTP system improves the potential for the biopharmaceutical industry to get products to patients more quickly.     

Enhancing the functionality of a microscale bioreactor system as an industrial process development tool for mammalian perfusion culture

Without a scale-down model for perfusion, high resource demand makes cell line screening or process development challenging, therefore, potentially successful cell lines or perfusion processes are unrealized and their ability untapped. We present here the refunctioning of a high-capacity microscale system that is typically used in fed-batch process development to allow perfusion operation utilizing in situ gravity settling and automated sampling. In this low resource setting, which involved routine perturbations in mixing, pH and dissolved oxygen concentrations, the specific productivity and the maximum cell concentration were higher than 3.0 × 10⁶ mg/cell/day and 7 × 10 ⁷ cells/ml, respectively, across replicate microscale perfusion runs conducted at one vessel volume exchange per day. A comparative analysis was conducted at bench scale with vessels operated in perfusion mode utilizing a cell retention device. Neither specific productivity nor product quality indicated by product aggregation (6%) was significantly different across scales 19 days after inoculation, thus demonstrating this setup to be a suitable and reliable platform for evaluating the performance of cell lines and the effect of process parameters, relevant to perfusion mode of culturing.     

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.     

The performance of a scaled down industrial disc stack centrifuge with a reduced feed material requirement

A method is described for the scale-down of a disc stack centrifuge which reduces the number of separating discs and also the liquid and solid hold-up of the centrifuge bowl. This is to enable a reduced volume of process material to be used for study of clarification. Scale-down is achieved in stages using a series of interlocking inserts to suit particular applications. Maximum scale-down gives a 76% reduction in the separation area and a bowl volume reduction of 70%. Separation performance of the full stack machine and scale-down variants is compared using the grade efficiency concept. Polyvinyl acetate and bakers' yeast homogenate particle suspensions are used for the comparison. The grade efficiency curves produced by the scale-down variants closely follow the curves for the full stack machine. This resulted in supernatants of the same volume size distribution and concentration when using scale-down methodology to mimic the full scale operation.

     

Prelude to rational scale-up of penicillin production: a scale-down study

Penicillin is one of the best known pharmaceuticals and is also an important member of the β-lactam antibiotics. Over the years, ambitious yields, titers, productivities, and low costs in the production of the β-lactam antibiotics have been stepwise realized through successive rounds of strain improvement and process optimization. Penicillium chrysogenum was proven to be an ideal cell factory for the production of penicillin, and successful approaches were exploited to elevate the production titer. However, the industrial production of penicillin faces the serious challenge that environmental gradients, which are caused by insufficient mixing and mass transfer limitations, exert a considerably negative impact on the ultimate productivity and yield. Scale-down studies regarding diverse environmental gradients have been carried out on bacteria, yeasts, and filamentous fungi as well as animal cells. In accordance, a variety of scale-down devices combined with fast sampling and quenching protocols have been established to acquire the true snapshots of the perturbed cellular conditions. The perturbed metabolome information stemming from scale-down studies contributed to the comprehension of the production process and the identification of improvement approaches. However, little is known about the influence of the flow field and the mechanisms of intracellular metabolism. Consequently, it is still rather difficult to realize a fully rational scale-up. In the future, developing a computer framework to simulate the flow field of the large-scale fermenters is highly recommended. Furthermore, a metabolically structured kinetic model directly related to the production of penicillin will be further coupled to the fluid flow dynamics. A mathematical model including the information from both computational fluid dynamics and chemical reaction dynamics will then be established for the prediction of detailed information over the entire period of the fermentation process and thereby for the optimization of penicillin production, and subsequently also benefiting other fermentation products.     

Scale-down/scale-up studies leading to improved commercial beer fermentation

Scale-up/scale-down techniques are vital for successful and safe commercial-scale bioprocess design and operation. An example is given in this review of recent studies related to beer production. Work at the bench scale shows that brewing yeast is not compromised by mechanical agitation up to 4.5 W/kg; and that compared with fermentations mixed by CO(2) evolution, agitation ≥ 0.04 W/kg is able to reduce fermentation time by about 20%. Work at the commercial scale in cylindroconical fermenters shows that, without mechanical agitation, most of the yeast sediments into the cone for about 50% of the fermentation time, leading to poor temperature control. Stirrer mixing overcomes these problems and leads to a similar reduction in batch time as the bench-scale tests and greatly reduces its variability, but is difficult to install in extant fermenters. The mixing characteristics of a new jet mixer, a rotary jet mixer, which overcomes these difficulties, are reported, based on pilot-scale studies. This change enables the advantages of stirring to be achieved at the commercial scale without the problems. In addition, more of the fermentable sugars are converted into ethanol. This review shows the effectiveness of scale-up/scale-down studies for improving commercial operations. Suggestions for further studies are made: one concerning the impact of homogenization on the removal of vicinal diketones and the other on the location of bubble formation at the commercial scale.     

An industrial perspective on bioreactor scale-down: what we can learn from combined large-scale bioprocess and model fluid studies

For industrial bioreactor design, operation, control and optimization, the scale-down approach is often advocated to efficiently generate data on a small scale, and effectively apply suggested improvements to the industrial scale. In all cases it is important to ensure that the scale-down conditions are representative of the real large-scale bioprocess. Progress is hampered by limited detailed and local information from large-scale bioprocesses. Complementary to real fermentation studies, physical aspects of model fluids such as air-water in large bioreactors provide useful information with limited effort and cost. Still, in industrial practice, investments of time, capital and resources often prohibit systematic work, although, in the end, savings obtained in this way are trivial compared to the expenses that result from real process disturbances, batch failures, and non-flyers with loss of business opportunity. Here we try to highlight what can be learned from real large-scale bioprocess in combination with model fluid studies, and to provide suitable computation tools to overcome data restrictions. Focus is on a specific well-documented case for a 30-m(3) bioreactor. Areas for further research from an industrial perspective are also indicated.     

The use of laboratory centrifugation studies to predict performance of industrial machines: studies of shear-insensitive and shear-sensitive materials

A method for using a bench-top centrifuge is described in order to mimic the recovery performance of an industrial-scale centrifuge, in this case a continuous-flow disc stack separator. Recovery performance was determined for polyvinyl acetate particles and for biological process streams of yeast cell debris and protein precipitates. Recovery of polyvinyl acetate particles was found to be well predicted for these robust particles. The laboratory centrifugation scale-down technique again predicted the performance of the disc stack centrifuge for the recovery of yeast cell debris particles although there was some suggestion of over-prediction at high levels of debris recovery due to the nature of any cell debris aggregates present. The laboratory centrifuge scale-down technique also proved to be an important investigative probe into the extent of shear-induced breakup of shear-sensitive protein precipitate aggregates during recovery in continuous high speed centrifuges. Such breakup can lead to over 10-fold reduction in separator capacity.     

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.     

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.