Sustaining an efficient and effective CHO cell line development platform by incorporation of 24‐deep well plate screening and multivariate analysis

Efficient and effective cell line screening is paramount toward a successful biomanufacturing program. Here we describe the implementation of 24‐deep well plate (24‐DWP) screening of CHO lines as part of the cell line development platform at AbbVie. Incorporation of this approach accelerated the identification of the best candidate lines for process development. In an effort to quantify and predict process performance comparability, we compared cell culture performance in and in shake flasks, for a panel of Chinese Hamster Ovary cell lines expressing a monoclonal antibody. The results in 24‐DWP screening showed reduced growth profiles, but comparable viability profiles. Slow growers in 24‐DWP achieved the highest productivity improvement upon scaling‐up to shake flasks. Product quality of the protein purified from shake flasks and 24‐DWP were also compared. The 24‐DWP culture conditions were found to influence the levels of acidic species, reduce the G0 N‐glycan species, and increase the high‐mannose N‐glycan species. Nevertheless, the identification of undesirable profiles is executed consistently with the scaled‐up culture. We further employed multivariate data analysis to capture differences depending on the two scales and we could demonstrate that cell line profiles were adequately clustered, regardless of the vessel used for the development. In conclusion, the 24‐DWP platform was reasonably predictive of the parameters crucial for upstream process development activities, and has been adapted as part of the AbbVie cell line development platform. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:175–186, 2018     

Equalizing growth in high‐throughput small scale cultivations via precultures operated in fed‐batch mode

An often underestimated problem when working with different clones in microtiter plates and shake flask screenings is the non‐parallel and non‐equal growth of batch cultures. These growth differences are caused by variances of individual clones regarding initial biomass concentration, lag‐phase or specific growth rate. Problems arising from unequal growth kinetics are different induction points in expression studies or uneven cultivation periods at the time of harvest. Screening for the best producing clones of a library under comparable conditions is thus often impractical or even impossible. A new approach to circumvent the problem of unequal growth kinetics of main cultures is the application of fed‐batch mode in precultures in microtiter plates and shake flasks. Fed‐batch operation in precultures is realized through a slow‐release system for glucose. After differently growing cultures turn to glucose‐limited growth, they all consume the same amount of glucose due to the fixed feed profile of glucose provided by the slow‐release system. This leads to equalized growth. Inherent advantages of this method are that it is easy to use and requires no additional equipment like pumps. This new technique for growth equalization in high‐throughput cultivations is simulated and verified experimentally. The growth of distinctly inoculated precultures in microtiter plates and shake flasks could be equalized for different microorganisms such as Escherichia coli and Hansenula polymorpha. Biotechnol. Bioeng. 2009;103: 1095–1102. © 2009 Wiley Periodicals, Inc.     

Predictive tool for recombinant protein production in Escherichia coli shake-flask cultures using an on-line monitoring system

As Escherichia coli (E. coli) is well defined with respect to its genome and metabolism, it is a favored host organism for recombinant protein production. However, many processes for recombinant protein production run under suboptimal conditions caused by wrong or incomplete information from an improper screening procedure, because appropriate on-line monitoring systems are still lacking. In this study, the oxygen transfer rate (OTR), determined on-line in shake flasks by applying a respiration activity monitoring system (RAMOS) device, was used to characterize the metabolic state of the recombinant organisms. Sixteen clones of E. coli SCS1 with foreign gene sequences, encoding for different target proteins, were cultivated in an autoinduction medium, containing glucose, lactose, and glycerol, to identify relationships between respiration activity and target protein production. All 16 clones showed a remarkably different respiration activity, biomass, and protein formation under induced conditions. However, the clones could be classified into three distinct types, and correlations could be made between OTR patterns and target protein production. For two of the three types, a decrease of the target protein was observed, after the optimal harvest time had passed. The acquired knowledge was used to modify the autoinduction medium to increase the product yield. Additional 1.5 g/L glucose accelerated the production process for one clone, shifting the time point of the maximal product yield from 24 to 17 h. For another clone, lactose addition led to higher volumetric product yields, in fact 25 and 38% more recombinant protein for 2 and 6 g/L additional lactose, respectively.     

Automation of cell line development

An automated platform for development of high producing cell lines for biopharmaceutical production has been established in order to increase throughput and reduce development costs. The concept is based on the Cello robotic system (The Automation Partnership) and covers screening for colonies and expansion of static cultures. In this study, the glutamine synthetase expression system (Lonza Biologics) for production of therapeutic monoclonal antibodies in Chinese hamster ovary cells was used for evaluation of the automation approach. It is shown that the automated procedure is capable of producing cell lines of equal quality to the traditionally generated cell lines in terms of colony detection following transfection and distribution of IgG titer in the screening steps. In a generic fed-batch evaluation in stirred tank bioreactors, IgG titers of 4.7 and 5.0 g/L were obtained for best expressing cell lines. We have estimated that the number of completed cell line development projects can be increased up to three times using the automated process without increasing manual workload, compared to the manual process. Correlation between IgG titers obtained in early screens and titers achieved in fed-batch cultures in shake flasks was found to be poor. This further implies the benefits of utilizing a high throughput system capable of screening and expanding a high number of transfectants. Two concentrations, 56 and 75 μM, of selection agent, methionine sulphoximine (MSX), were applied to evaluate the impact on the number of colonies obtained post transfection. When applying selection medium containing 75 μM MSX, fewer low producing transfectants were obtained, compared to cell lines selected with 56 μM MSX, but an equal number of high producing cell lines were found. By using the higher MSX concentration, the number of cell line development projects run in parallel could be increased and thereby increasing the overall capacity of the automated platform process. A. Salmén and K. Lindgren contributed equally to the work.     

Real-time monitoring of shake flask fermentation and off gas using triple disposable noninvasive optical sensors

Bioprocess development is a data-driven process requiring a large number of experiments to be conducted under varying conditions. Small-scale upstream bioprocess development is often performed in shake flasks because they are inexpensive and can be operated in parallel. However, shake flasks are often not equipped to accurately monitor critical process parameters such as pH, dissolved oxygen, and CO2 concentrations. Therefore, there is no definitive information on oxygen supply of growing cells, CO2 formation, and pH changes. Here we describe several shake flask fermentations where all three parameters are monitored by disposable noninvasive optical sensors. The sensitive element of these sensors is a thin, luminescent patch affixed inside the flask. Small electronic devices for excitation and fluorescence detection are positioned outside the shake flask for noninvasive monitoring. By measuring the process parameters throughout the course of the E. coli fermentations, we obtain information that is not routinely available in shake flask fermentations. For example, for cultures with only a few millimeters liquid depth, oxygen limitation can occur at relatively low agitation speeds. Under certain conditions oscillations in dissolved oxygen can occur. An increase in shaker speed and a decrease in culture volume can increase the oxygen availability and reduce the duration of oxygen limitation.     

Optimization of cultivation conditions in spin tubes for Chinese hamster ovary cells producing erythropoietin and the comparison of glycosylation patterns in different cultivation vessels

This article describes the optimization of cultivation factor settings, that is the shaking rate and working volume in 50 mL spin tubes for a Chinese hamster ovary cell line expressing recombinant human α‐erythropoietin, using a response D‐optimal surface method. The main objectives of the research were, firstly, to determine a setting in which the product titer and product quality attributes in spin tubes are equivalent to those in 250 mL shake flasks in a seven day batch and, secondly, to find a setting in which the product titer is maximal. The model for product titer prediction as a function of shaking rate and working volume in the defined design space was successfully applied to the optimization of cultivation conditions in spin tubes for the tested cell line. Subsequently, validation experiments were carried out simultaneously in spin tubes, shake flasks and bench scale bioreactors to compare cell culture performance parameters such as growth, productivity and product quality attributes in the form of isoform profiles and glycan antennarity structures. The results of the experiments showed that similar cell culture performance and product quality could be achieved in spin tubes when compared to shake flasks. Additionally, bioreactor titers could be reproduced in spin tubes at high shaking rates and low working volumes, but with differing product quality. Cultivation at lower shaking rates in spin tubes and shake flasks produced a glycoprotein with a product quality slightly comparable to that from bioreactors, but with titers being only two thirds. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

A protocol to transfer a fed-batch platform process into semi-perfusion mode: The benefit of automated small-scale bioreactors compared to shake flasks as scale-down model

Continuous processes such as perfusion processes can offer advantages compared to fed-batch or batch processes in bio-processing: improved product quality (e.g. for labile products), increased product yield, and cost savings. In this work, a semi-perfusion process was established in shake flasks and transferred to an automated small-scale bioreactor by daily media exchange via centrifugation based on an existing fed-batch process platform. At first the development of a suitable medium and feed composition, the glucose concentration required by the cells and the cell-specific perfusion rate were investigated in shake flasks as the conventional scale-down system. This lead to an optimized process with a threefold higher titer of 10 g/L monoclonal antibody compared to the standard fed-batch. To proof the suitability and benefit as a small-scale model, the established semi-perfusion process was transferred to an automated small-scale bioreactor with improved pH and dissolved oxygen control. The average specific productivity improved from 24.16 pg/(c*d) in the fed-batch process and 36.04 pg/c*d in the semi-perfusion shake flask to 38.88 pg/(c*d) in the semi-perfusion process performed in the controlled small-scale bioreactor, thus illustrating the benefits resulting from the applied semi-perfusion approach, especially in combination with controlled DO and pH settings. © 2019 The Authors. Biotechnology Progress published by Wiley Periodicals, Inc. on behalf of American Institute of Chemical Engineers Biotechnol. Prog., 35: e2757, 2019.     

Advances in clone selection using high‐throughput bioreactors

Effective clone selection is a crucial step toward developing a robust mammalian cell culture production platform. Currently, clone selection is done by culturing cells in well plates and picking the highest producers. Ideally, clone selection should be done in a stirred tank bioreactor as this would best replicate the eventual production environment. The actual number of clones selected for future evaluation in bioreactors at bench‐scale is limited by the scale‐up and operational costs involved. This study describes the application of miniaturized stirred high‐throughput bioreactors (35 mL working volume; HTBRs) with noninvasive optical sensors for clone screening and selection. We investigated a method for testing several subclones simultaneously in a stirred environment using our high throughput bioreactors (up to 12 clones per HTBR run) and compared it with a traditional well plate selection approach. Importantly, it was found that selecting clones solely based on results from stationary well plate cultures could result in the chance of missing higher producing clones. Our approach suggests that choosing a clone after analyzing its performance in a stirred bioreactor environment is an improved method for clone selection. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Advanced microscale bioreactor system: a representative scale-down model for bench-top bioreactors

In recent years, several automated scale-down bioreactor systems have been developed to increase efficiency in cell culture process development. ambr™ is an automated workstation that provides individual monitoring and control of culture dissolved oxygen and pH in single-use, stirred-tank bioreactors at a working volume of 10–15 mL. To evaluate the ambr™ system, we compared the performance of four recombinant Chinese hamster ovary cell lines in a fed-batch process in parallel ambr™, 2-L bench-top bioreactors, and shake flasks. Cultures in ambr™ matched 2-L bioreactors in controlling the environment (temperature, dissolved oxygen, and pH) and in culture performance (growth, viability, glucose, lactate, Na⁺, osmolality, titer, and product quality). However, cultures in shake flasks did not show comparable performance to the ambr™ and 2-L bioreactors.     

Micro-bioreactors for fed-batch fermentations with integrated online monitoring and microfluidic devices

In this study an array of micro-bioreactors based on the format of 48-well microtiter plates (MTP) is presented. The process parameters pH-value and biomass are monitored online by a combination of different sensors, the biolector measurement technology and conductance measurements. A microfluidic device dispenses two fluids individually into each well for controlling the pH-value of fermentations. The micro-bioreactor consists of four wells and two reservoirs. In each well a polyimide foil with platinum electrodes for conductance measurements is integrated. The microfluidic device is fabricated using softlithographic techniques and utilizes pneumatically actuated microvalves. The device is able to dispense volumes below 5nl. Finally, fermentations of Escherichia coli are carried out in the micro-bioreactor system. During the fermentation, the pH-value is measured optically and the biomass development is monitored by the scattered light signal. Meanwhile, the pH-value is controlled by dispensing sodium hydroxide and phosphoric acid. This micro-bioreactor demonstrates the possibility of online monitored and pH-controlled fermentations in micro-scale. The pH-value in the uncontrolled culture varies within the range of 6.46-8.83 whereas the pH-value in the controlled cultures can be kept within 6.85-7.07. This results in an increase in biomass in the pH-controlled culture compared to the nearly completely inhibited pH-uncontrolled culture.     

In situ pH maintenance for mammalian cell cultures in shake flasks and tissue culture flasks

pH in animal cell cultures decreases due to production of metabolites like lactate. pH control via measurement and base addition is not easily possible in small‐scale culture formats like tissue‐culture flasks and shake flasks. A hydrogel‐based system is reported for in situ pH maintenance without pH measurement in such formats, and is demonstrated to maintain pH between 6.8 and 7.2 for a suspension CHO cell line in CD CHO medium and between 7.3 and 7.5 for adherent A549 cells in DMEM:F12 containing 10% FBS. This system for pH maintenance, along with our previous report of hydrogels for controlled nutrient delivery in shake flasks can allow shake flasks to better mimic bioreactor‐based fed batch operation for initial screening during cell line and process development for recombinant protein production in mammalian cells. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Effective temperature shift strategy development and scale confirmation for simultaneous optimization of protein productivity and quality in Chinese hamster ovary cells

Temperature shifts to lower culture temperatures are frequently employed in the manufacturing of protein therapeutics in mammalian cells to improve productivity, viability, or quality attributes. The direction and extent to which a temperature shift affects productivity and quality may vary depending on the expression host and characteristics of the expressed protein. We demonstrated here that two Chinese hamster ovary (CHO) clones expressing different human monoclonal antibodies responded differently to a temperature shift despite sharing a common parental CHO cell line. Within a single CHO line, we observed a nonlinear response to temperature shift. A moderate shift to 35°C significantly decreased final titer relative to the unshifted control while a larger shift to 32°C significantly increased final titer by 25%. Therefore, we proposed a systematic empirical approach to assess the utility of a temperature shift for faster implementation during process development. By testing multiple shift parameters, we identified optimum shift conditions in shake flasks and successfully translated findings to benchtop bioreactors and 1,000-L bioreactor scale. Significant differences in final antibody titer and charge variants were observed with temperature shift increments as small as Δ1.5°C. Acidic charge variants decreased monotonically with decreasing shift temperature in both cell lines; however, final antibody titer required simultaneous optimization of shift day and temperature. Overall, we were able to show that a systematic approach to identify temperature shift parameters at small scales is useful to optimize protein production and quality for efficient and confident translation to large-scale production.     

Process performance of parallel bioreactors for batch cultivation of Streptomyces tendae

Batch cultivations of the nikkomycin Z producer Streptomyces tendae were performed in three different parallel bioreactor systems (milliliter-scale stirred-tank reactors, shake flasks and shaken microtiter plate) in comparison to a standard liter-scale stirred-tank reactor as reference. Similar dry cell weight concentrations were measured as function of process time in stirred-tank reactors and shake flasks, whereas only poor growth was observed in the shaken microtiter plate. In contrast, the nikkomycin Z production differed significantly between the stirred and shaken bioreactors. The measured product concentrations and product formation kinetics were almost the same in the stirred-tank bioreactors of different scale. Much less nikkomycin Z was formed in the shake flasks and MTP cultivations, most probably due to oxygen limitations. To investigate the non-Newtonian shear-thinning behavior of the culture broth in small-scale bioreactors, a new and simple method was applied to estimate the rheological behavior. The apparent viscosities were found to be very similar in the stirred-tank bioreactors, whereas the apparent viscosity was up to two times increased in the shake flask cultivations due to a lower average shear rate of this reactor system. These data illustrate that different engineering characteristics of parallel bioreactors applied for process development can have major implications for scale-up of bioprocesses with non-Newtonian viscous culture broths.     

Chinese hamster ovary K1 host cell enables stable cell line development for antibody molecules which are difficult to express in DUXB11‐derived dihydrofolate reductase deficient host cell

Therapeutic monoclonal antibodies (mAb) are often produced in Chinese hamster ovary (CHO) cells. Three commonly used CHO host cells for generating stable cell lines to produce therapeutic proteins are dihydrofolate reductase (DHFR) positive CHOK1, DHFR‐deficient DG44, and DUXB11‐based DHFR deficient CHO. Current Genentech commercial full‐length antibody products have all been produced in the DUXB11‐derived DHFR‐deficient CHO host. However, it has been challenging to develop stable cell lines producing an appreciable amount of antibody proteins in the DUXB11‐derived DHFR‐deficient CHO host for some antibody molecules and the CHOK1 host has been explored as an alternative approach. In this work, stable cell lines were developed for three antibody molecules in both DUXB11‐based and CHOK1 hosts. Results have shown that the best CHOK1 clones produce about 1 g/l for an antibody mAb1 and about 4 g/l for an antibody mAb2 in 14‐day fed batch cultures in shake flasks. In contrast, the DUXB11‐based host produced ～0.1 g/l for both antibodies in the same 14‐day fed batch shake flask production experiments. For an antibody mAb3, both CHOK1 and DUXB11 host cells can generate stable cell lines with the best clone in each host producing ～2.5 g/l. Additionally, studies have shown that the CHOK1 host cell has a larger endoplasmic reticulum and higher mitochondrial mass. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:980–985, 2013     

Enhanced production of l‐lactic acid by Lactobacillus thermophilus SRZ50 mutant generated by high‐linear energy transfer heavy ion mutagenesis

The aim of this study was to improve l ‐lactic acid production of Lactobacillus thermophilus SRZ50. For this purpose, high efficient heavy‐ion mutagenesis technique was performed using SRZ50 as the original strain. To enhance the screening efficiency for high yield l ‐lactic acid producers, a scale‐down from shake flask to microtiter plate was developed. The results showed that 24‐well U‐bottom MTPs could well alternate shake flasks for L. thermophilus cultivation as a scale‐down tool due to its a very good comparability to the shake flasks. Based on this microtiter plate screening method, two high l ‐lactic acid productivity mutants, A59 and A69, were successfully screened out, which presented, respectively, 15.8 and 16.2% higher productivities than that of the original strain. Based on fed‐batch fermentation, the A69 mutant can accumulate 114.2 g/L l ‐lactic acid at 96 h. Hence, the proposed traditional microbial breeding method with efficient high‐throughput screening assay was proved to be an appropriate strategy to obtain lactic acid‐overproducing strain.     

Microscale Perfusion-Based Cultivation for Pichia pastoris Clone Screening Enables Accelerated and Optimized Recombinant Protein Production Processes

Pichia pastoris has emerged in the past years as a promising host for recombinant protein and biopharmaceutical production. In the establishment of high cell density fed-batch biomanufacturing, screening phase and early bioprocess development (based on microplates and shake flasks) still represent a bottleneck due to high-cost and time-consuming procedures as well as low experiment complexity. In the present work, a screening protocol developed for P. pastoris clone selection is implemented in a multiplexed microfluidic device with 15 μL cultivation chambers able to operate in perfusion mode and monitor dissolved oxygen content in the culture in a non-invasive way. The setup allowed us to establish carbon-limited conditions and evaluate strain responses to different input variables. Results from micro-scale perfusion cultures are then compared with 1L fed-batch fermentation. The best producer in terms of titer and productivity is rapidly identified after 12 h from inoculation and the results confirmed by lab-scale fermentation. Moreover, the physiological analyses of the strains under different conditions suggested how more complex experimental conditions are achievable despite the relatively easy, straight-forward, and cost-effective experimental setup. Implementation and standardization of these micro-scale protocols could reduce the demand for lab-scale bioreactor cultivations thus accelerating the development of protein production processes.     

Novel micro‐bioreactor high throughput technology for cell culture process development: Reproducibility and scalability assessment of fed‐batch CHO cultures

With increasing timeline pressures to get therapeutic and vaccine candidates into the clinic, resource intensive approaches such as the use of shake flasks and bench‐top bioreactors may limit the design space for experimentation to yield highly productive processes. The need to conduct large numbers of experiments has resulted in the use of miniaturized high‐throughput (HT) technology for process development. One such high‐throughput system is the SimCell? platform, a robotically driven, cell culture bioreactor system developed by BioProcessors Corp. This study describes the use of the SimCell? micro‐bioreactor technology for fed‐batch cultivation of a GS‐CHO transfectant expressing a model IgG4 monoclonal antibody. Cultivations were conducted in gas‐permeable chambers based on a micro‐fluidic design, with six micro‐bioreactors (MBs) per micro‐bioreactor array (MBA). Online, non‐invasive measurement of total cell density, pH and dissolved oxygen (DO) was performed. One hundred fourteen parallel MBs (19 MBAs) were employed to examine process reproducibility and scalability at shake flask, 3‐ and 100‐L bioreactor scales. The results of the study demonstrate that the SimCell? platform operated under fed‐batch conditions could support viable cell concentrations up to least 12 × 10⁶ cells/mL. In addition, both intra‐MB (MB to MB) as well as intra‐MBA (MBA to MBA) culture performance was found to be highly reproducible. The intra‐MB and ‐MBA variability was calculated for each measurement as the coefficient of variation defined as CV (%) = (standard deviation/mean) × 100. The % CV values for most intra‐MB and intra‐MBA measurements were generally under 10% and the intra‐MBA values were slightly lower than those for intra‐MB. Cell growth, process parameters, metabolic and protein titer profiles were also compared to those from shake flask, bench‐top, and pilot scale bioreactor cultivations and found to be within ±20% of the historical averages. Biotechnol. Bioeng. 2010; 106: 57–67. © 2010 Wiley Periodicals, Inc.     

Integration of cell line and process development to overcome the challenge of a difficult to express protein

This case study addresses the difficulty in achieving high level expression and production of a small, very positively charged recombinant protein. The novel challenges with this protein include the protein's adherence to the cell surface and its inhibitory effects on Chinese hamster ovary (CHO) cell growth. To overcome these challenges, we utilized a multi‐prong approach. We identified dextran sulfate as a way to simultaneously extract the protein from the cell surface and boost cellular productivity. In addition, host cells were adapted to grow in the presence of this protein to improve growth and production characteristics. To achieve an increase in productivity, new cell lines from three different CHO host lines were created and evaluated in parallel with new process development workflows. Instead of a traditional screen of only four to six cell lines in bioreactors, over 130 cell lines were screened by utilization of 15 mL automated bioreactors (AMBR) in an optimal production process specifically developed for this protein. Using the automation, far less manual intervention is required than in traditional bench‐top bioreactors, and much more control is achieved than typical plate or shake flask based screens. By utilizing an integrated cell line and process development incorporating medium optimized for this protein, we were able to increase titer more than 10‐fold while obtaining desirable product quality. Finally, Monte Carlo simulations were performed to predict the optimal number of cell lines to screen in future cell line development work with the goal of systematically increasing titer through enhanced cell line screening. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1201–1211, 2015     

Advances in shaking technologies

Shaking bioreactors are the most frequently used reactor system for screening and process optimization on a small scale. Their success can be attributed to their simple and functional design, which make shaking systems suitable for a large number of cost-efficient parallel experiments. Recently reported findings for oxygen transfer, power input, out-of-phase operation, hydromechanical stress and mixing in shaken bioreactors are summarized in this article. Novel monitoring techniques for the control of culture conditions in shake flasks and microtiter plates are described. The methods for characterizing culture conditions and the novel online measurement techniques that are summarized in this article can be utilized to tap the full potential of shaking reactor systems.     

用克隆池PCR法筛选小麦-簇毛麦易位系6VS/6AL基因组TAC文库获得抗病基因候选克隆

用根据抗病基因保守区设计的一对简并性引物,从小麦－簇毛麦易位系6VS／6AL cDNA中PCR扩增获得一个具有抗病基因核苷酸结合位点（Nucleotide binding site,NBS）结构特点的DNA片段克隆N7。从小麦－簇毛麦易位系6VS／6AL基因组TAC（Transformation-competent artificial chromosome,TAC）文库的22块96孔板提取所有2112个克隆池（每个池含约1000个克隆）的质粒,再根据N7的核苷酸序列设计一对特异引物,用克隆池PCR（pooled PCR)法经分级筛选从文库中获得一个阳性克隆。以N7为探针,通过Southern杂交证实了该TAC克隆为真正含有抗病候选基因的克隆。研究结果表明克隆池PCR法对克隆数目巨大的基因组文库的筛选很有效。