Application of high‐throughput mini‐bioreactor system for systematic scale‐down modeling,process characterization,and control strategy development

High‐throughput systems and processes have typically been targeted for process development and optimization in the bioprocessing industry. For process characterization, bench scale bioreactors have been the system of choice. Due to the need for performing different process conditions for multiple process parameters, the process characterization studies typically span several months and are considered time and resource intensive. In this study, we have shown the application of a high‐throughput mini‐bioreactor system viz. the Advanced Microscale Bioreactor (ambr15^TM), to perform process characterization in less than a month and develop an input control strategy. As a pre‐requisite to process characterization, a scale‐down model was first developed in the ambr system (15 mL) using statistical multivariate analysis techniques that showed comparability with both manufacturing scale (15,000 L) and bench scale (5 L). Volumetric sparge rates were matched between ambr and manufacturing scale, and the ambr process matched the pCO₂ profiles as well as several other process and product quality parameters. The scale‐down model was used to perform the process characterization DoE study and product quality results were generated. Upon comparison with DoE data from the bench scale bioreactors, similar effects of process parameters on process yield and product quality were identified between the two systems. We used the ambr data for setting action limits for the critical controlled parameters (CCPs), which were comparable to those from bench scale bioreactor data. In other words, the current work shows that the ambr15^TM system is capable of replacing the bench scale bioreactor system for routine process development and process characterization. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1623–1632, 2015     

A novel scale-down mimic of perfusion cell culture using sedimentation in an automated microbioreactor (SAM)

Continuous upstream processing in mammalian cell culture for recombinant protein production holds promise to increase product yield and quality. To facilitate the design and optimization of large-scale perfusion cultures, suitable scale-down mimics are needed which allow high-throughput experiments to be performed with minimal raw material requirements. Automated microbioreactors are available that mimic batch and fed-batch processes effectively but these have not yet been adapted for perfusion cell culture. This article describes how an automated microbioreactor system (ambr15) can be used to scale-down perfusion cell cultures using cell sedimentation as the method for cell retention. The approach accurately predicts the viable cell concentration, in the range of about 1 × 10⁷ cells/mL for a human cell line, and cell viability of larger scale cultures using a hollow fiber based cell retention system. While it was found to underpredict cell line productivity, the method accurately predicts product quality attributes, including glycosylation profiles, from cultures performed in bioreactors with working volumes between 1 L and 1,000 L. The spent media exchange method using the ambr15 was found to predict the influence of different media formulations on large-scale perfusion cultures in contrast to batch and chemostat experiments performed in the microbioreactor system. The described experimental setup in the microbioreactor allowed an 80-fold reduction in cell culture media requirements, half the daily operator time, which can translate into a cost reduction of approximately 2.5-fold compared to a similar experimental setup at bench scale.     

High‐throughput miniaturized bioreactors for cell culture process development: Reproducibility,scalability, and control

Decreasing the timeframe for cell culture process development has been a key goal toward accelerating biopharmaceutical development. Advanced Microscale Bioreactors (ambr?) is an automated micro‐bioreactor system with miniature single‐use bioreactors with a 10–15 mL working volume controlled by an automated workstation. This system was compared to conventional bioreactor systems in terms of its performance for the production of a monoclonal antibody in a recombinant Chinese Hamster Ovary cell line. The miniaturized bioreactor system was found to produce cell culture profiles that matched across scales to 3 L, 15 L, and 200 L stirred tank bioreactors. The processes used in this article involve complex feed formulations, perturbations, and strict process control within the design space, which are in‐line with processes used for commercial scale manufacturing of biopharmaceuticals. Changes to important process parameters in ambr? resulted in predictable cell growth, viability and titer changes, which were in good agreement to data from the conventional larger scale bioreactors. ambr? was found to successfully reproduce variations in temperature, dissolved oxygen (DO), and pH conditions similar to the larger bioreactor systems. Additionally, the miniature bioreactors were found to react well to perturbations in pH and DO through adjustments to the Proportional and Integral control loop. The data presented here demonstrates the utility of the ambr? system as a high throughput system for cell culture process development. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:718–727, 2014     

An anti-apoptotic HEK293 cell line provides a robust and high titer platform for transient protein expression in bioreactors

ABSTRACT

HEK293 transient expression systems are used to quickly generate proteins for research and pre-clinical studies. With the aim of engineering a high-producing host that grows and transfects robustly in bioreactors, we deleted the pro-apoptotic genes Bax and Bak in an HEK293 cell line. The HEK293 Bax Bak double knock-out (HEK293 DKO) cell line exhibited resistance to apoptosis and shear stress. HEK293 DKO cells sourced from 2 L seed train bioreactors were most productive when a pH setpoint of 7.0, a narrow pH deadband of ±0.03, and a DO setpoint of 30% were used. HEK293 DKO seed train cells cultivated for up to 60 days in a 35 L bioreactor showed similar productivities to cells cultivated in shake flasks. To optimize HEK293 DKO transfection cultures, we first evaluated different pH and agitation parameters in ambr15 microbioreactors before scaling up to 10 L wavebag bioreactors. In ambr15 microbioreactors with a pH setpoint of 7.0, a wide pH deadband of ±0.3, and an agitation of 630 rpm, HEK293 DKO transient cultures yielded antibody titers up to 650 mg/L in 7 days. The optimal ambr15 conditions prompted us to operate the 10 L wavebag transfection without direct pH control to mimic the wide pH deadband ranges. The HEK293 DKO transfection process produces high titers at all scales tested. Combined, our optimized HEK293 DKO 35 L bioreactor seed train and 10 L high titer transient processes support efficient, large-scale recombinant protein production for research studies.     

Process development of human multipotent stromal cell microcarrier culture using an automated high‐throughput microbioreactor

Microbioreactors play a critical role in process development as they reduce reagent requirements and can facilitate high‐throughput screening of process parameters and culture conditions. Here, we have demonstrated and explained in detail, for the first time, the amenability of the automated ambr15 cell culture microbioreactor system for the development of scalable adherent human mesenchymal multipotent stromal/stem cell (hMSC) microcarrier culture processes. This was achieved by first improving suspension and mixing of the microcarriers and then improving cell attachment thereby reducing the initial growth lag phase. The latter was achieved by using only 50% of the final working volume of medium for the first 24 h and using an intermittent agitation strategy. These changes resulted in >150% increase in viable cell density after 24 h compared to the original process (no agitation for 24 h and 100% working volume). Using the same methodology as in the ambr15, similar improvements were obtained with larger scale spinner flask studies. Finally, this improved bioprocess methodology based on a serum‐based medium was applied to a serum‐free process in the ambr15, resulting in >250% increase in yield compared to the serum‐based process. At both scales, the agitation used during culture was the minimum required for microcarrier suspension, N_JS. The use of the ambr15, with its improved control compared to the spinner flask, reduced the coefficient of variation on viable cell density in the serum containing medium from 7.65% to 4.08%, and the switch to serum free further reduced these to 1.06–0.54%, respectively. The combination of both serum‐free and automated processing improved the reproducibility more than 10‐fold compared to the serum‐based, manual spinner flask process. The findings of this study demonstrate that the ambr15 microbioreactor is an effective tool for bioprocess development of hMSC microcarrier cultures and that a combination of serum‐free medium, control, and automation improves both process yield and consistency. Biotechnol. Bioeng. 2017;114: 2253–2266. © 2017 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Efficient high‐throughput biological process characterization: Definitive screening design with the Ambr250 bioreactor system

The burgeoning pipeline for new biologic drugs has increased the need for high‐throughput process characterization to efficiently use process development resources. Breakthroughs in highly automated and parallelized upstream process development have led to technologies such as the 250‐mL automated mini bioreactor (ambr250?) system. Furthermore, developments in modern design of experiments (DoE) have promoted the use of definitive screening design (DSD) as an efficient method to combine factor screening and characterization. Here we utilize the 24‐bioreactor ambr250? system with 10‐factor DSD to demonstrate a systematic experimental workflow to efficiently characterize an Escherichia coli (E. coli) fermentation process for recombinant protein production. The generated process model is further validated by laboratory‐scale experiments and shows how the strategy is useful for quality by design (QbD) approaches to control strategies for late‐stage characterization. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1388–1395, 2015     

High throughput automated microbial bioreactor system used for clone selection and rapid scale‐down process optimization

High throughput automated fermentation systems have become a useful tool in early bioprocess development. In this study, we investigated a 24 x 15 mL single use microbioreactor system, ambr 15f, designed for microbial culture. We compared the fed‐batch growth and production capabilities of this system for two Escherichia coli strains, BL21 (DE3) and MC4100, and two industrially relevant molecules, hGH and scFv. In addition, different carbon sources were tested using bolus, linear or exponential feeding strategies, showing the capacity of the ambr 15f system to handle automated feeding. We used power per unit volume (P/V) as a scale criterion to compare the ambr 15f with 1 L stirred bioreactors which were previously scaled‐up to 20 L with a different biological system, thus showing a potential 1,300 fold scale comparability in terms of both growth and product yield. By exposing the cells grown in the ambr 15f system to a level of shear expected in an industrial centrifuge, we determined that the cells are as robust as those from a bench scale bioreactor. These results provide evidence that the ambr 15f system is an efficient high throughput microbial system that can be used for strain and molecule selection as well as rapid scale‐up. © 2017 The Authors Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 34:58–68, 2018     

Segmented linear modeling of CHO fed‐batch culture and its application to large scale production

We describe a systematic approach to model CHO metabolism during biopharmaceutical production across a wide range of cell culture conditions. To this end, we applied the metabolic steady state concept. We analyzed and modeled the production rates of metabolites as a function of the specific growth rate. First, the total number of metabolic steady state phases and the location of the breakpoints were determined by recursive partitioning. For this, the smoothed derivative of the metabolic rates with respect to the growth rate were used followed by hierarchical clustering of the obtained partition. We then applied a piecewise regression to the metabolic rates with the previously determined number of phases. This allowed identifying the growth rates at which the cells underwent a metabolic shift. The resulting model with piecewise linear relationships between metabolic rates and the growth rate did well describe cellular metabolism in the fed‐batch cultures. Using the model structure and parameter values from a small‐scale cell culture (2 L) training dataset, it was possible to predict metabolic rates of new fed‐batch cultures just using the experimental specific growth rates. Such prediction was successful both at the laboratory scale with 2 L bioreactors but also at the production scale of 2000 L. This type of modeling provides a flexible framework to set a solid foundation for metabolic flux analysis and mechanistic type of modeling. Biotechnol. Bioeng. 2017;114: 785–797. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Scale‐down of the inactivated polio vaccine production process

The anticipated increase in the demand for inactivated polio vaccines resulting from the success in the polio eradication program requires an increase in production capacity and cost price reduction of the current inactivated polio vaccine production processes. Improvement of existing production processes is necessary as the initial process development has been done decades ago. An up‐to‐date lab‐scale version encompassing the legacy inactivated polio vaccine production process was set‐up. This lab‐scale version should be representative of the large scale, meaning a scale‐down model, to allow experiments for process optimization that can be readily applied. Initially the separate unit operations were scaled‐down at setpoint. Subsequently, the unit operations were applied successively in a comparative manner to large‐scale manufacturing. This allows the assessment of the effects of changes in one unit operation to the consecutive units at small‐scale. Challenges in translating large‐scale operations to lab‐scale are discussed, and the concessions that needed to be made are described. The current scale‐down model for cell and virus culture (2.3‐L) presents a feasible model with its production scale counterpart (750‐L) when operated at setpoint. Also, the current scale‐down models for the DSP unit operations clarification, concentration, size exclusion chromatography, ion exchange chromatography, and inactivation are in agreement with the manufacturing scale. The small‐scale units can be used separately, as well as sequentially, to study variations and critical product quality attributes in the production process. Finally, it is shown that the scale‐down unit operations can be used consecutively to prepare trivalent vaccine at lab‐scale with comparable characteristics to the product produced at manufacturing scale. Biotechnol. Bioeng. 2013; 110: 1354–1365. © 2012 Wiley Periodicals, Inc.     

Ultra scale‐down approach to correct dispersive and retentive effects in small‐scale columns when predicting larger scale elution profiles

Ultra scale‐down approaches represent valuable methods for chromatography development work in the biopharmaceutical sector, but for them to be of value, scale‐down mimics must predict large‐scale process performance accurately. For example, one application of a scale‐down model involves using it to predict large‐scale elution profiles correctly with respect to the size of a product peak and its position in a chromatogram relative to contaminants. Predicting large‐scale profiles from data generated by small laboratory columns is complicated, however, by differences in dispersion and retention volumes between the two scales of operation. Correcting for these effects would improve the accuracy of the scale‐down models when predicting outputs such as eluate volumes at larger scale and thus enable the efficient design and operation of subsequent steps. This paper describes a novel ultra scale‐down approach which uses empirical correlations derived from conductivity changes during operation of laboratory and pilot columns to correct chromatographic profiles for the differences in dispersion and retention. The methodology was tested by using 1 mL column data to predict elution profiles of a chimeric monoclonal antibody obtained from Protein A chromatography columns at 3 mL laboratory‐ and 18.3 L pilot‐scale. The predictions were then verified experimentally. Results showed that the empirical corrections enabled accurate estimations of the characteristics of larger‐scale elution profiles. These data then provide the justification to adjust small‐scale conditions to achieve an eluate volume and product concentration which is consistent with that obtained at large‐scale and which can then be used for subsequent ultra scale‐down operations. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2009     

Optimization of medium with perfusion microbioreactors for high density CHO cell cultures at very low renewal rate aided by design of experiments

A novel approach of design of experiment (DoE) is developed for the optimization of key substrates of the culture medium, amino acids, and sugars, by utilizing perfusion microbioreactors with 2 mL working volume, operated in high cell density continuous mode, to explore the design space. A mixture DoE based on a simplex-centroid is proposed to test multiple medium blends in parallel perfusion runs, where the amino acids concentrations are selected based on the culture behavior in presence of different amino acid mixtures, and using targeted specific consumption rates. An optimized medium is identified with models predicting the culture parameters and product quality attributes (G0 and G1 level N-glycans) as a function of the medium composition. It is then validated in runs performed in perfusion microbioreactor in comparison with stirred-tank bioreactors equipped with alternating tangential flow filtration (ATF) or with tangential flow filtration (TFF) for cell separation, showing overall a similar process performance and N-glycosylation profile of the produced antibody. These results demonstrate that the present development strategy generates a perfusion medium with optimized performance for stable Chinese hamster ovary (CHO) cell cultures operated with very high cell densities of 60 × 10⁶ and 120 × 10⁶ cells/mL and a low cell-specific perfusion rate of 17 pL/cell/day, which is among the lowest reported and is in line with the framework recently published by the industry.     

Elevated pCO2 affects the lactate metabolic shift in CHO cell culture processes

The shift from lactate production to consumption in CHO cell metabolism is a key event during cell culture cultivations and is connected to increased culture longevity and final product titers. However, the mechanisms controlling this metabolic shift are not yet fully understood. Variations in lactate metabolism have been mainly reported to be induced by process pH and availability of substrates like glucose and glutamine. The aim of this study was to investigate the effects of elevated pCO₂ concentrations on the lactate metabolic shift phenomena in CHO cell culture processes. In this publication, we show that at elevated pCO₂ in batch and fed‐batch cultures, the lactate metabolic shift was absent in comparison to control cultures at lower pCO₂ values. Furthermore, through metabolic flux analysis we found a link between the lactate metabolic shift and the ratio of NADH producing and regenerating intracellular pathways. This ratio was mainly affected by a reduced oxidative capacity of cultures at elevated pCO₂. The presented results are especially interesting for large‐scale and perfusion processes where increased pCO₂ concentrations are likely to occur. Our results suggest, that so far unexplained metabolic changes may be connected to increased pCO₂ accumulation in larger scale fermentations. Finally, we propose several mechanisms through which increased pCO₂ might affect the cell metabolism and briefly discuss methods to enable the lactate metabolic shift during cell cultivations.     

Enhanced process understanding and multivariate prediction of the relationship between cell culture process and monoclonal antibody quality

This work investigates the insights and understanding which can be deduced from predictive process models for the product quality of a monoclonal antibody based on designed high‐throughput cell culture experiments performed at milliliter (ambr‐15^®) scale. The investigated process conditions include various media supplements as well as pH and temperature shifts applied during the process. First, principal component analysis (PCA) is used to show the strong correlation characteristics among the product quality attributes including aggregates, fragments, charge variants, and glycans. Then, partial least square regression (PLS1 and PLS2) is applied to predict the product quality variables based on process information (one by one or simultaneously). The comparison of those two modeling techniques shows that a single (PLS2) model is capable of revealing the interrelationship of the process characteristics to the large set product quality variables. In order to show the dynamic evolution of the process predictability separate models are defined at different time points showing that several product quality attributes are mainly driven by the media composition and, hence, can be decently predicted from early on in the process, while others are strongly affected by process parameter changes during the process. Finally, by coupling the PLS2 models with a genetic algorithm first the model performance can be further improved and, most importantly, the interpretation of the large‐dimensioned process–product‐interrelationship can be significantly simplified. The generally applicable toolset presented in this case study provides a solid basis for decision making and process optimization throughout process development. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1368–1380, 2017     

Perfusion seed cultures improve biopharmaceutical fed‐batch production capacity and product quality

Volumetric productivity and product quality are two key performance indicators for any biopharmaceutical cell culture process. In this work, we showed proof‐of‐concept for improving both through the use of alternating tangential flow perfusion seed cultures coupled with high‐seed fed‐batch production cultures. First, we optimized the perfusion N‐1 stage, the seed train bioreactor stage immediately prior to the production bioreactor stage, to minimize the consumption of perfusion media for one CHO cell line and then successfully applied the optimized perfusion process to a different CHO cell line. Exponential growth was observed throughout the N‐1 duration, reaching >40 × 10⁶ vc/mL at the end of the perfusion N‐1 stage. The cultures were subsequently split into high‐seed (10 × 10⁶ vc/mL) fed‐batch production cultures. This strategy significantly shortened the culture duration. The high‐seed fed‐batch production processes for cell lines A and B reached 5 g/L titer in 12 days, while their respective low‐seed processes reached the same titer in 17 days. The shortened production culture duration potentially generates a 30% increase in manufacturing capacity while yielding comparable product quality. When perfusion N‐1 and high‐seed fed‐batch production were applied to cell line C, higher levels of the active protein were obtained, compared to the low‐seed process. This, combined with correspondingly lower levels of the inactive species, can enhance the overall process yield for the active species. Using three different CHO cell lines, we showed that perfusion seed cultures can optimize capacity utilization and improve process efficiency by increasing volumetric productivity while maintaining or improving product quality. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 30:616–625, 2014     

Development,characterization, and application of a 2‐Compartment system to investigate the impact of pH inhomogeneities in large‐scale CHO‐based processes

Large‐scale bioreactors for the production of monoclonal antibodies reach volumes of up to 25 000 L. With increasing bioreactor size, mixing is however affected negatively, resulting in the formation of gradients throughout the reactor. These gradients can adversely affect process performance at large scale. Since mammalian cells are sensitive to changes in pH, this study investigated the effects of pH gradients on process performance. A 2‐Compartment System was established for this purpose to expose only a fraction of the cell population to pH excursions and thereby mimicking a large‐scale bioreactor. Cells were exposed to repeated pH amplitudes of 0.4 units (pH 7.3), which resulted in decreased viable cell counts, as well as the inhibition of the lactate metabolic shift. These effects were furthermore accompanied by increased absolute lactate levels. Continuous assessment of molecular attributes of the expressed target protein revealed that subunit assembly or N‐glycosylation patterns were only slightly influenced by the pH excursions. The exposure of more cells to the same pH amplitudes further impaired process performance, indicating this is an important factor, which influences the impact of pH inhomogeneity. This knowledge can aid in the design of pH control strategies to minimize the effects of pH inhomogeneity in large‐scale bioreactors.     

Model‐assisted identification of metabolic engineering strategies for Jatropha curcas lipid pathways

Efficient approaches to increase plant lipid production are necessary to meet current industrial demands for this important resource. While Jatropha curcas cell culture can be used for in vitro lipid production, scaling up the system for industrial applications requires an understanding of how growth conditions affect lipid metabolism and yield. Here we present a bottom‐up metabolic reconstruction of J. curcas supported with labeling experiments and biomass characterization under three growth conditions. We show that the metabolic model can accurately predict growth and distribution of fluxes in cell cultures and use these findings to pinpoint energy expenditures that affect lipid biosynthesis and metabolism. In addition, by using constraint‐based modeling approaches we identify network reactions whose joint manipulation optimizes lipid production. The proposed model and computational analyses provide a stepping stone for future rational optimization of other agronomically relevant traits in J. curcas.