Cell Culture and Upstream Processing
Lewis Ho (Bioreactor Sciences)
Case Studies of hERG Membrane Protein and Japanese Encephalitis Virus Production Using a New, Innovative, Moving-Bed Bioreactor
Human erythroblast-transformation– related gene (ERG) membrane protein has gained great interest as a target for drug discovery. A doubled expression of the hERG gene was induced under nutrient limitations. The protein is membrane bound, so whole-cell recovery from the carriers is required. HEK293 cells were used. A Japanese encephalitis virus (JEV) vaccine has long been the only reliable solution to prevent this deadly disease. However, the virus is unstable. Postinfection conditions are critical for optimal virus production and recovery. We use those two case studies to illustrate a new innovative moving-bed (MB) bioreactor’s unsurpassed versatility and functionality to accommodate specific desired conditions and accomplish the entire upstream process in one unit. The mobility of the moving matrix bed allows for the least amount of volume required for seeding, yielding high seed density for increased cell attachment. That feature results in high virus infection and DNA transfection efficiency. The MB system facilitates a principle similar to roller bottles, intermittently exposing and submersing cells to air and medium to achieve maximum oxygenation with near minimal shear while eliminating foaming. The MB sets a new precedent in performing the mechanism of cell detachment and recovery in place.
Nihal Almuraikhi (Imperial College London)
A Novel System to Enhance Production of Erythroid Cells from Human Induced Pluripotent Stem Cells Using HIF-1α
Blood shortage is a major global concern because of a significant imbalance
in donations relative to transfusion demand, which encourages researchers to develop substitutes for donated blood using pluripotent stem cells. A rising excitement is surrounding use of human induced pluripotent stem (iPS) cells to provide patient-specific cells for cellular therapy. Their capability to differentiate into any cell type also can be exploited in erythroid cell production. Erythropoiesis is the process of making erythrocytes that can be enhanced by hypoxia-inducible factors 1-α (HIF-1α), a transcription factor known to facilitate cellular adaptation to hypoxia by overexpressing specific genes and stimulating many metabolic processes, including erythropoiesis, and angiogenesis. We established a novel protocol to generate erythroid cells from human iPS cells using HIF-1α as a key enhancer. In addition to the standard cytokine cocktail for erythroid induction (Epo, SCF, FLT3, TPO, IL3, and IL6), we used other growth factors — BMP4, VEGF, FGF — and 5% serum. Supplementing cells in a two-dimensional (2D) culture system with our novel optimized concentrations of that cocktail under hypoxic condition showed the highest yield erythroid markers, mainly CD235a. Further maturation of those cells is required to achieve fully mature and functional red–blood-cell (RBC) phenotype. These results must be supported by detection of Rh type and ABO grouping to ensure the presence of RBC antigens. Eventually, after optimizing an enucleation protocol, a globin detection and O2 dissociation curve also must be made to ensure hemoglobin functionality (incomplete work). Considering all of this, the ultimate aim of our study is efficient production of mature and functional RBCs in vitro from patient-specific iPS cells using HIF-1α to facilitate erythroid progenitor maturation and proliferation.
Michael Biksacky (Flownamics)
An Automated Bioreactor Sampling Solution for Assuring On-line PAT Analytical Fidelity
In the current bioprocess environment, a significant unmet need remains for automated, precise, real-time analysis of important culture process parameters such as nutrients, metabolites, and various cell measurements. Transforming traditional off-line analytical methods into effective, automated, on-line process analytical technology (PAT) platforms requires rapid and precise analysis that performs as good as or better than the manual off-line analytical method. An automated, on-line bioreactor sampling system was evaluated for its ability to preserve the analytical integrity of different integrated, third-party, off- line analyzers typically used for culture process monitoring. The performance evaluation encompassed a broad range of analytes and measurement ranges while comparing automated on-line analysis with the analyzer’s respective manual, off-line analytical method. In short, the analytical fidelity of each integrated analyzer was preserved by the automated on-line sampling system. That demonstrated a reliable, automated, on-line process analytical technology (PAT) solution for real-time bioreactor culture monitoring and control.
Ashley Fisher (Flownamics)
Seg-Flow Automated Nutrient Monitoring and Continuous-Feed Platform for Precise Nutrient Feed Control
Fed-batch culture processes are commonly used for production of biotherapeutic and industrial commodities. A widely implemented nutrient feed-control strategy for both cell culture and microbial fermentation processes is direct feedback control, by which nutrient feed is directly controlled by the nutrient (e.g., glucose, concentration). However, different iterations of that feeding approach may not be conducive to culture growth and productivity. To optimize culture performance, the means of feeding substrate is just as important as (if not more so than) maintaining desired media substrate levels. Flownamics has developed an automated feedback control platform using a continuous-feed strategy based on a culture’s real-time nutrient consumption rate. This integrated platform consists of a Seg-Flow automated on-line sampling system with a nutrient analyzer and feed pump. The Seg-Flow system was evaluated for its ability to precisely control media glucose concentrations for both low– and high–consumption- rate culture simulation models. Results show that the integrated system precisely controlled media glucose concentrations at prescribed levels through real-time calculation of glucose consumption rate along with continuous feeding.
Wen Wang (Massachusetts Institute of Technology)
High-Throughput Screening for Mammalian Cell Clones
The biopharmaceutical industry has grown quickly over the past decade, creating a $100 billion market worldwide with the approval of recombinant proteins, monoclonal antibodies (MAbs), and nucleic-acid–based drugs. Chinese hamster ovary (CHO) cells are the most widely used mammalian cell systems for transfection, expression, and large- scale recombinant protein production because of their advantages in accurate glycosylation for in vitro production of natural proteins. Significant improvement in recombinant protein production has been made in recent CHO cell culture technology (leading to expression titers ≤10 g/L) to meet high market demands with stable and high producers while optimizing culture processes. However, rapid selection for high clone production is still needed. We applied a new screening concept for on-line and in situ high- throughput screening of mammalian cells. We used a CHO cell line producing anti- her2 as our model system. By measuring the key parameters of the cell culture system on line, we could predict cell-line performance and identify high producers without performing invasive assays. The impact of this research will be significant to upstream bioprocesses. In cell-line development, product titer is a crucial parameter for clone selection. Our method requires no external reporter, and it is a noninvasive, liquid-based assay. We need monitor only basic information to calculate product quantity. High and low producers can be easily and quickly identified on-line and in situ using automated handling and preprogramming.
Nick Kohlstrom (Eppendorf)
Perfusion CHO Cell Culture in a BioBLU 5p Packed-Bed Single- Use Vessel
The market for secreted recombinant protein therapeutics, including blockbuster drugs in the form of humanized monoclonal antibodies (hMAbs), has become a multibillion-dollar industry with continued growth expected. A cost-effective method of production
uses packed-bed vessels operated under perfusion conditions. Maximum cell densities thus achieved are typically much higher than for suspension cell culture or microcarrier-based adherent cell culture. Protein harvest can be carried out continuously, providing unparalleled product yields. We used a BioBLU 5p packed-bed single-use vessel to conduct perfusion CHO cell culture producing a secreted hMAb. The vessel (preloaded with Fibra-Cel disks) was controlled by a New Brunswick CelliGen BLU benchtop bioreactor system. It was inoculated at an initial cell density of 0.3 × 106 cells/mL for a 14-day perfusion cell culture with a working volume of 3.75 L. Glucose, lactate and hMAb concentration were monitored daily. We used the glucose consumption rate to estimate cell density in the packed-bed vessel. After 12 days, the culture reached a peak cell density of about 10 × 106 cells/mL.
Khandaker Siddiquee (Eppendorf)
Large-Scale Production of Human Mesenchymal Stem Cells in BioBLU 5c Single-Use Vessels
Stem-cell–based regenerative medicine has great potential to revolutionize human disease treatment. Among different stem cell platforms, mesenchymal stem cells (MSCs) represent high potential as evidenced by clinical trial activities: Currently, more than 400 clinical trials using MSCs are registered at clinicaltrials.gov. Although successful in vitro expansion of MSCs is well established, clinical-scale production of MSCs remains a bottleneck, potentially limiting their immediate clinical applications should some ongoing trials receive FDA approval. We demonstrate the success of commercial-scale culture of human adipose-derived mesenchymal stem cells (AdMSCs) using an industrial single-use vessel at 3.75-L scale (working volume). The vessel offers a precision- controlled environment for ideal growth of stem cells under simulated physiological conditions. Stem cells and culture media were monitored, analyzed, and controlled to allow for production of AdMSCs in large quantities with maintenance of healthy stem-cell properties as evidenced by marker and differentiation assays performed at the end of the culture. Furthermore, with clinical relevance in mind, every cell culture step — from T-flask to shake flask to bioreactor vessel — was conducted strictly using single-use consumables.
David Laidlaw (Kuhner Shaker)
Development and Characterization of an Orbitally Shaken 12-L Bioreactor Suitable for Mammalian and Microcarrier Cultures
The pharmaceutical market demands single-use bioreactor alternatives to rocking bags for culturing mammalian cells in the 2-L to 10-L scales. Orbitally shaken vessels expose cells to low shear stress, making these systems suitable for cultivation of shear-sensitive organisms. We characterized a disposable 12-L culture vessel that was constructively extended to a bioreactor through integration of an on-line monitoring system (pH and dissolved oxygen, DO). We investigated the influence of those integrated sensors and microcarriers on scale-up parameters. Our data will support all users of shaken bioreactors in optimizing their specific cell-cultivation conditions.
Yamuna Dasarathy (Pall Life Sciences)
Efficient Separation of Antibody Light Chains from Bispecific Antibody Monomer Using Mixed-Mode Sorbents
Bispecific antibodies are considered as a promising class of next-generation therapeutic molecules. In coming years, a growing number of such products are expected to arrive on the market. Purification of bispecific antibodies presents different challenges from those of MAbs, and chromatographic sorbents will have to answer those specific requirements. We investigated the use of mixed-mode sorbents as a capture step for purification of a bispecific antibody, with specific focus on separating the abundant antibody fragments (light chains) from the monomeric form. Three mixed-mode sorbents were evaluated for their performance in antibody light- chain elimination. Our data indicate that two out of those three provided efficient removal of antibody light chains with selective elution of antibody from a column as fragments were retained on the resin. Up to 97% pure monomers were recovered after only one purification step using mixed-mode sorbents, whereas the initial monomer purity was about 30%. Satisfactory recovery and efficient CHO host-cell protein removal (>1 log) were obtained. Altogether, these data reveal that mixed-mode chromatography is a powerful tool for addresing future challenges in purification of the growing bispecific antibody class of biomolecules.
Olivier Berteau (Charter Medical)
Continuous CHO Cell Culture Perfusion Platform for Process Development and Manufacturing
Bioreactor design is a complex engineering task that requires fine- tuning of hardware design and operational configuration to enable cultured organisms to achieve a desired function. Before large-scale commercial manufacturing can be implemented, it is typically necessary to scale up a bioreactor process during process development. But conventional scale-up techniques are fraught with technical challenges and financial risks. We present a perfusion bioreactor system operating a plurality of modular perfused bioreactors in parallel to produce and maintain a biological cell culture using a commercially available CHO cell line. To prevent contamination and facilitate segregation of particular peripheral bioreactors, the distributed system is configured such that one-way fluid communication is established from the central bioreactor to each peripheral bioreactor while maintaining fluid isolation among those bioreactors. In accordance with one exemplary nonlimiting embodiment, a distributed perfusion bioreactor system is provided for producing and maintaining a continuous biological cell culture. Modularity of the peripheral bioreactors enables scale-up–free design of an exemplary bioreactor system that eliminates the disadvantages of conventional scale-up. Modularity of the peripheral bioreactors enables detection of process deviations and contamination, with easy and efficient segregation or restart of an affected peripheral bioreactor branch without disrupting the entire system. A distributed bioreactor arrangement can maintain cell culture continuously for extended periods.
Qiang Fei (National Bioenergy Center)
Sensitivity Analysis of BioGTL Process
Considering the relatively low price of natural gas and increasing demands of
liquid transportation fuels, attention has begun to turn to novel biocatalysts for conversion of natural gas into diesel fuel (Bio-GTL). A technoeconomic analysis (TEA) was performed for an integrated fermentation process converting natural gas to microbial lipids and catalytic upgrading lipids to diesel blend stocks. A sensitivity analysis was carried out based on identified key parameters such as plant capacity, process technology (yields and rates), productivity, and raw material cost. We also compare production of natural-gas–derived diesel fuel with other fuels produced from different platforms.
Stacey Willard (Eppendorf)
Microalgae Culture Using the DASGIP PBR4 Module for Illumination with a New Brunswick CelliGen 310 Bioreactor
The number of bioprocess applications for microalgae has increased in recent years, particularly in biofuel production. The global biofuels market is predicted to reach US$100 billion in 2014. Microalgae bioprocess applications could expand into a multibillion-dollar sector of that growing global market. The combination of a New Brunswick CelliGen 310 benchtop autoclavable bioreactor and DASGIP LED Illumination system creates a bioprocess setup capable of supporting high-density microalgal growth. Using a stand-alone DASGIP PBR4 module, analysts can control LED illumination spectra and intensities for optimal support of all types of chlorophylls and carotenoids. For this study (in which high- density culture of ≤1.5 × 107 cells/mL was achieved), the unicellular marine alga Dunaliella tertiolecta was used.
Bin Li (Enppendorf)
High–Cell-Density Fermentation of Escherichia coli Using the New Brunswick BioFlo 115 Benchtop Fermentor
E. coli is a Gram-negative bacterium with a long history in laboratory and industrial processes due to its ease of manipulation and well-understood genome. It is widely cultured under aerobic conditions. High–cell-density cultivation of E. coli is a powerful technique for production of recombinant proteins. Indeed, 30% of FDA-approved biopharmaceuticals on the market are produced using E. coli. The easy-to-use New Brunswick BioFlo 115 benchtop bioprocess system from Eppendorf has built-in controls for operation as a microbial fermentor and features Rushton impellers; a high-speed, direct-drive motor; and an optional thermal-mass flow controller (TMFC) with automatic gas mixing. This system is ideal for research and development laboratories, universities, teaching facilities, testing laboratories, and more. The poster presents a successful example of high-density fermentation of E. coli using a BioFlo 115 fermentor with 2-L (working volume) heat-blanketed glass vessel. E. coli cultivation reached a high optical density (OD600) value of 140 at 11 hours without optimized fermentation medium or conditions.
Recovery and Purification
Martin Hoffman (Biotechflow)
The Renaissance of Expanded-Bed Adsorption?
Two decades after its initial development, expanded-bed adsorption (EBA) chromatography fell out of favor in bioprocessing. Proposed improvements in process efficiency and obtaining high yields from unclarified feed stocks were compromised by column design and process run failures. Those were in part caused by blocked meshes, lipid breakthrough, and demand for high-buffer volumes producing high eluting volumes. Current media optimize dynamic binding capacity (DBC) within the expanded bed. Coupled with an improved inlet design, increased bed densities are achieved that permit high flow rates (450 cm/hr) and throughputs. Direct product capture reduces unit costs (by 66%) as well as labor costs and process times. Comparisons with packed-bed chromatography result in higher product yields and a 43% reduction in buffer volume. Fully scalable, CGMP EBA column design improvements prevent vortex formation with ultrasound technology to enable contactless monitoring of process dynamics and flow rate by feedback to the skid. Both factors maximize expanded-bed stability. Reduced column footprint and height requirements increase versatility. In recent years, step changes in incubation techniques, media design, column technology, and sensor applications have enabled proven improvements in EBA process efficiency and operations. The initial promise now may be considered as surpassed, and a renaissance of EBA chromatography should be proposed.
Ron Massicotte (Genentech/Roche)
Improving Safety and Efficiency in a Demanding Pilot-Plant Environment
We will illustrate and explain several improvements that have enhanced safety and efficiency in a busy pilot-plant environment.
Oliver Hardick (University College London)
Nanofibers for High-Productivity Downstream Processing
Payers worldwide are becoming ever more price sensitive, and successful exploitation of emerging and biosimilar markets will require rethinking current production strategies to refocus on cost of goods, flexibility, and developability. Chromatography is moving from a one- size-fits-all, bead-based process to one of increasing product stratification as new entrants launch technically specified products to address specific process issues.
Nancy Ngo (Bio-Rad Laboratories)
Direct Quantification of Residual Host-Cell DNA
Many therapeutic proteins and vaccines are manufactured using bacterial and mammalian host cells. Manufacturing processes are prone to leaving biological impurities from these cells, such as host-cell DNA (HCD). The presence of HCD in drug substances poses safety concerns, so it must be removed to ensure product quality and safety. Regulatory agencies such as the US Food and Drug Administration (FDA) and the World Health Organization (WHO) have provided guidelines defining acceptable levels of HCD allowed in final drug products. The upper limits stated by the FDA and WHO are 100 pg/dose and 10 ng/dose, respectively. Therefore, the method of quantifying residual HCD and monitoring DNA clearance should be highly sensitive to meet those requirements. Here, we introduce a highly precise and sensitive method for residual HCD quantification without DNA extraction using droplet digital PCR (ddPCR). We created a panel of test matrices to simulate various process intermediates and analyzed the samples using ddPCR. Spiked-in DNA can be fully recovered even in complex matrices with high precision and femtogram-level sensitivity. Results from our study clearly show the effectiveness of ddPCR in detecting residual HCD by a direct method without a need for DNA extraction.
Quality and Analytical
Moria Shimoni (VAYU sense)
VAYU Meter: Biomonitoring Device
VAYU sense’s rapid screening device, the “VAYU meter,” was developed for detection of living cells and microorganisms in growth medium. it measures CO2 metabolic gas concentrations produced during cell respiration and proliferation. The VAYU meter enables continuous metabolic gas detection for high- sensitivity bioprocess control, growth- rate monitoring, and bioconcentration. By means of infrared absorption, this device allows in situ detection of cells and microorganisms in fermentors or bioreactors directly from reactor exhaust, without a need for opening, sampling, or incubating. This device can be applied for quantitative analysis and estimating the level of biological activity of cells and microorganisms in biopharmaceutical processes. Fermentation processes of most important and complex pharmaceuticals require precise culture-base growth. From the early stages of commercial protein production, handling of cultures is subject to challenges. Commercial production of products at large scale depends greatly on the stable maintenance of cell levels. To optimize biotech production processes and improve yields and economics, companies need better, precise, real-time process understanding and improved equipment use. The VAYU meter is a real- time process-status monitoring solution that can accurately indicate relative reaction conditions. This high-resolution, continuous-detection system is successful in detecting cells and microorganisms’ concentration.
Dana Kinzlmaier (EMD Millipore)
Use of Mobius MIX Vessels with Protein Solutions: Impact of Impeller Flow and Particle Shedding on Protein Turbidity and Aggregation
Increasing implementation of single- use equipment and assemblies in biopharmaceutical processes has produced flexible facilities that enable
fast batch-to-batch and product-to- product turnaround times. However, it has also led to new questions about how materials of construction and system functionalities might affect drug products being made. Users considering a move to single-use mixing systems for buffer preparation typically want information about particulate count and generation and extractables/leachables (identity and levels) as well as guidance on effective mixing protocols to ensure robust dissolution of buffer components. But when considering the same mixing systems for protein pools, additional concerns arise over the potential effects of impeller action and particulates on protein quality and stability. To gain better understanding regarding the Mobius MIX family for use with protein pools, we performed a study to determine whether we could detect a change in protein quality during mixing and whether that change could be correlated to a mixing parameter. In addition, we evaluated whether generated particulates affect short-term protein stability. [end symbol]
This is just a sample of the posters that will be on display in the exhibit hall. These early arrivals will be joined by more that likewise provide cutting-edge science, techniques, and expertise to improve existing methods and processes while reducing time and cost. BioProcess International (in partnership with the BPI Conference) is proud to present the Best Poster Award at the 2014 BioProcess International Conference and Exhibition. Posters will be reviewed and judged by the editors with members of the BPI Editorial Advisory Board. Two winning posters — one from academia/industry and one from a supplier — will be announced at the conference and published in BPI’s November 2014 Interactive Poster Hall supplement. Posters, complete with prerecorded audiocast presentation, will also be posted online for a full year at bioprocessintl.com/ posters.