Key Factors for Successful Plasmid Production

BioFlo® 320 – Universal controller for cell culture and microbiology research The Smarter Solution 2 BioFlo® 320 BioFlo® 320 3 Highly Evolved The BioFlo 320 seamlessly combines form and function in one state of the art package regardless of whether your process includes cell culture or fermentation, autoclavable or single-use bioreactors. A robust industrial design, intelligent sensors, Ethernet connectivity, and enhanced software capabilities are only a few of the features that set it apart from the competition. Combined with a sincere commitment to quality, the BioFlo 320 truly is the premium choice in bench-scale bioprocess control systems. Ease of Use > Autoclavable vessels and our comprehensive portfolio of BioBLU® Single-Use Bioreactors provide process customization > Eppendorf exclusive packed-bed and cell-lift impeller designs for continuous and perfusion processes > Thermal mass flow controllers for sparge and overlay gas can be upgraded in the field > Universal gas control strategy for both microbial and cell culture applications removes process limitations > Industry standard Ethernet communication for multi-unit control of up to eight systems Application Driven > Industrial design featuring stainless steel front, back, and utility panels > Left- and right-handed orientations to maximize lab space efficiency > Hemispherical vessel nest design for minimum footprint > Highly configurable gas flow control - Up to 5 configurable Mass Flow Controllers (TMFC) for Overlay and Sparge Scalability > Extensive working volume range of 400 mL – 40 L on a single control platform > High-powered direct and magnetic drive motor assemblies > Up to six integrated pumps capable of operating in variable speed mode > The combination of the BioFlo 320 with the BioFlo 720 provides a perfect connection between R&D and pilot/production suites Risk mitigation > Monitor and control the process directly at the large touch-screen or with our Eppendorf SCADA software > Alarm functions automatically stop all running loops if needed > Automatic recognition of the connected digital sensors and installed TMFCs > Robust protection of sensitive electronic components (IP68-rated connections on utility panel and IP22-rated connection for power entry) > Universal connections for analog or digital Mettler Toledo® ISM® sensors reduce sensor complexity Small footprint... big impact From R&D laboratories to pilot-scale production facilities, space is an important factor when selecting the right equipment. The BioFlo 320 offers flexibility, better control, and maximum functionality while occupying a fraction of the valuable lab space of similar systems. This means greater efficiency and productivity at a lower operating cost for your lab. The next generation of our BioFlo 320 bioprocess control system combines the benefits of a classical industrial design with the power of our improved BioFlo control software. Developed to be used in cGMP regulated environments, Emerson® and Eppendorf have partnered to develop seamless communication between the DeltaVTM distributed control system and the BioFlo 320 bioprocess control system. Our open communication protocol exposes controller information allowing it to be integrated into broader features and functions of the DeltaV platform, simplifying tech transfer, scale-up, and recipe sharing in bioprocess research and process development. Application Driven Designed for cGMP environments 4 BioFlo® 320 Single-use bioreactors: 0.40 L – 40 L Vessel material: Glass or Single-Use Process control: Cell culture or Microbiology Autoclavable bioreactors: 0.6 L – 10.5 L Control up to eight units from a single user interface High-powered direct and magnetic drive motor assemblies; capable of clockwise and counterclockwise rotation Ultra-compact design with vessel nest for minimum footprint; left- and right-handed orientations to maximize lab space Unique variable/ fixed speed frontmounted pumps capable of clockwise and counterclockwise rotation Industrial, brushed stainless steel panels provide enhanced corrosion resistance Sixteen interchangeable autoclavable vessels and the comprehensive line of BioBLU Single-Use Bioreactors Four universal connections for analog or digital Mettler Toledo ISM sensors Interchangeable TMFC drawers for sparge and overlay gas flexibility BioBLU® Single-Use Bioreactors BioFlo® 320 5 Scalability The perfect connection from R&D to production Seamless transition of your process from 400 mL to 40 L and beyond. In combination with the BioFlo 720 bioprocess control system, we designed a bioreactor control platform that supports one of the widest range of single-use solutions, allowing the scale-up of your process to up to 2,000 L. The updated and improved BioFlo software ensures a consistent user experience across all BioFlo control systems and comes with new features to improve your process. > Compatible with 400 mL – 40 L BioBLU Single-Use Bioreactors, including the BioBLU 5p, the first single-use bioreactor to utilize the exclusive packed-bed impeller > Built-in optical pH sensor technology > Vessel and exhaust heat blanket connections provide precise temperature control and exhaust gas treatment > Bench-scale single-use bioreactors specifically designed for microbial fermentation > Rigid-walled, stirred-tank design provides many advantages over single-use bag design > Eliminates potential for tears, pits, and folds during installation > Single-layer polymer removes both uncertainty for leachable and extractable data and the need for unnecessary preventative actions, like pre-process media wash Application flexibility > Suitable for the use in all labs, from academia through pilot-scale production > Batch, fed-batch, perfusion, and continuous processes > Universal control for mammalian, stem cell, insect, microbial, plant, and algae cultures > Secreted product, vaccine, and monoclonal antibody production > Biofuels research and manufacturing > Scale-up and scale-down modeling > Suspension or adherent cultures > Micro-aerobic, anaerobic, exothermic fermentation processes > Specialized impellers for low-shear and zero-shear process needs > Food and beverage > Fine chemical processes > Integrated Mettler Toledo Intelligent Sensor Management (ISM) platform > Universal connections for up to four analog or digital (ISM) sensors > pH: analog or digital (ISM) > DO: analog or digital (ISM) > Optical DO: digital (ISM) > Redox: analog or digital (ISM) > CO2 : digital (ISM) > Real-time sensor diagnostics anticipate sensor failure Full integration of bioprocess software and data into Emerson’s DeltaVTM system Eppendorf and Emerson have collaborated to integrate Emerson’s DeltaV™ distributed control system (DCS) with the BioFlo 320 bioprocess control system. > Full integration of bioprocess software and data into Emerson's DeltaV system > Simplified tech-transfer, scale-up, and recipe sharing in bioprocess research > Manage data and use the same automation systems throughout the product development process, from bioprocess research to manufacturing Intelligent Sensors 6 BioFlo® 320 Monitoring the controller and the process is key to successful process development. The BioFlo 320 software features screens, designed to provide you with the status of both. > Alarm functions automatically inform about the status of the process, with automatic loop shutdown if needed > Receive alarm notifications via email or text message directly to your mobile device > The diagnostic screen provides information on the hardware status and upcoming maintenance Risk Mitigation Parallel processing is key to efficiently optimizing a process or to producing reproducible results. Eight units of the BioFlo 320 bioprocess control system can be connected to each other and controlled by a single unit. The control software gives valuable insights into all processes, such as current process parameters, and the lifetime of connected digital sensors. Monitoring and control of critical system parameters BioFlo® 320 7 Ease of Use Advanced software making life easier Auto Calibrate > Automated calibration for all attached DO sensors at once > DO sensors can be run through an automatic calibration sequence reducing touch time and ensuring consistency > User specifies calibration scheme, process conditions, and zero-point method (electronic or N2 ) > System completes calibration when slope stabilizes The new release of the BioFlo software ensures a consistent user experience across all BioFlo control systems and comes with new features to improve your process. Develop your process with the BioFlo 320 and seamlessly scale-up from 50 L – 2,000 L with the BioFlo 720 bioprocess controller. New software features such as the Auto Calibrate and Scale Up Assist function simplify your process, mitigate risk, and help to save time. Parallel Control > Control eight units from a single user interface > Automatic gas mixing algorithms for simplified control (4-gas, 3-gas, O2 enrichment, N2 enrichment) > Ten-point cascade feature for sophisticated control strategies > Built-in elapsed fermentation timer for batch management > Trend display with up to twelve process values within a single view Scale Up Assist > Scale Up modeling software for the calculation of important process parameters > The integrated Scale Up Assist of the BioFlo software calculates all parameters based on either constant P/V or constant tip speed > Up to 3 different vessel sizes can be selected from > The software contains vessel specific data from the Eppendorf vessel portfolio and the ThermoTM Scientific HyPerformaTM 5:1 Single-Use Bioreactors (SUBs). Mettler Toledo® and ISM® are registered trademarks of Mettler Toledo AG, Switzerland. Watson-Marlow® is a registered trademark of Watson-Marlow Limited, UK. Emerson® is a registered trademark of Emerson Electric Co., USA. DeltaV™ is a trademark of Emerson Electric Co., USA. Thermo ScientificTM and HyPerformaTM are trademarks of Thermo Fisher Scientific Inc, USA. Eppendorf®, the Eppendorf Brand Design, and BioBLU® are registered trademarks of Eppendorf SE, Germany. DASware® is a registered trademark of DASGIP Information and Process Technology GmbH, Germany. BioFlo® and BioCommand® are registered trademarks of Eppendorf, Inc., USA. All rights reserved, including graphics and images. Copyright © 2022 by Eppendorf SE. Order No.: AN00111020/GB5/0.5T/0824/EBC/STEFF · Carbon neutrally printed in Germany www.eppendorf.com/BioFlo-320 Your local distributor: www.eppendorf.com/contact Eppendorf SE · Barkhausenweg 1 · 22339 Hamburg · Germany eppendorf@eppendorf.com · www.eppendorf.com Technical Data BioFlo 320 Specifications Control Station Dimensions (W x D x H) 40.6 x 40.6 x 66.0 cm (16 in x 16 x 26 in) Net weight 32 kg (70 lb) Touchscreen 38.1 cm (15 in) projected capacitive touchscreen Communication 2 x USB (software updates, serial communication) Ethernet (SCADA, IP Network) 3 x Analog Input/Output (defined as 4 – 20 mA or 0 – 5 V or 0 – 10 V) Utility Connection Requirement Electrical IEC (with regional plug types) 100 – 120/208 – 240 VAC, 50/60 Hz, 2270 VA, Single Phase Water Stainless steel quick-connect 10 psig (0.69 barg) Gas supply (Air, O2 , N2 , CO2 ) Push-connect Autoclavable Single-use 10 psig (0.69 barg) 6 psig (0.44 barg) Exhaust 0.5 psig (0.035 barg) Operating conditions 10 – 30 °C, up to 80 % RH, non-condensing Agitation Direct drive 1 L, 3 L: 25 – 1500 rpm 5 L, 10 L: 25 – 1200 rpm Magnetic drive (autoclavable vessels) 1 L, 3 L, or 5 L: 10 – 500 rpm 10 L: 10 – 150 rpm Magnetic drive (single-use vessels) BioBLU 1f: 10 – 1200 rpm; BioBLU 3f: 25 – 1200 BioBLU 1c: 10 – 500 rpm BioBLU 3c, 5c, 5p, 10c & 14c: 10 – 200 rpm BioBLU 50c: 10 – 150 rpm Temperature Water-jacketed 5 °C above coolant to 55 °C above ambient (80 °C max) Stainless steel dish-bottom 5 °C above coolant to 65 °C above ambient (90 °C max; 85 °C max for 10 L) Single-use 5 °C above ambient to 40 °C (60 °C max for BioBLU 1) Sensor type PT100 Gas supply Sparge 1, 3, or 4 TMFC; ring or micro-sparger Overlay 1 TMFC; headspace addition Sensors Communication Control range pH Analog or digital Mettler Toledo ISM 2 – 12 Optical pH Digital (Presens) 6 – 8 DO Analog or digital Mettler Toledo ISM 0 – 200 % Optical DO Digital Mettler Toledo ISM 0 – 200 % Redox Analog or digital Mettler Toledo ISM (-)2000 mV – (+)2000 mV CO2 Digital Mettler Toledo ISM 0 – 100 % Pumps Pump Head Variable Speed Fixed Speed Pumps 1, 2, & 3 Watson-Marlow 114DV 5 – 25 rpm 25 rpm (0 – 100 % Duty Cycle) Pump 4 Watson-Marlow 314D 20 – 100 rpm 100 rpm (0 – 100 % Duty Cycle) External pumps 1 & 2 Watson-Marlow 120U/DV 0.1 – 200 rpm N/A Specifications subject to change. Seamless Integration of Glucose Control using Raman Spectroscopy in CHO Cell Culture Célia Sanchez, Laure Pétillot, Fabrice Thomas, Charlotte Javalet Merck KGaA, Darmstadt, Germany 13 chemin du Vieux Chêne, 38240 Meylan, France Contact: bioprocess-experts@eppendorf.com APPLICATION NOTE No. 415 Abstract In the context of Process Analytical Technologies (PAT) implementation in the biopharmaceutical industry, Quality by Design (QbD) is being developed and widely implemented and used. In upstream processes, one compound of great interest to monitor is glucose, and specifically, being able to control its concentration during the process. Such a monitoring leads to process quality improvement, including glycosylation of the product of interest. In this study, a Raman analyzer has been successfully used to implement a feedback control loop in a CHO cell culture based on glucose concentration. The feedback control loop implied a direct OPC UA connectivity between the analyzer and the bioreactor control system The culture was fed with a complex feed containing glucose. As a result, glucose concentration was maintained steady for three days. The process performance remained similar to the ones of regular fed batch cultures and a noteworthy decrease in lactate production was observed. The process was completely automated for glucose concentration management and did not require any human intervention throughout the process. Introduction The Process Analytical Technology (PAT) and Quality by Design (QbD) guidelines, promoted by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) aims to support the idea that quality cannot be tested only into a product but must instead be deployed throughout design. Seamless integration of monitoring and control of analytical data into a bioprocess is crucial to understand a process and to overcome manufacturing challenges. One of the biggest challenges is the monitoring of quality attributes such as glycosylation. Important characteristics like stability and immunogenicity are affected by glycosylation. In order to receive regulatory approval, glycosylation is a Critical Quality Attribute (CQA) ensuring the safety and potency of biopharmaceutical products. Maintaining the glucose concentration steady is key for the control and optimization of processes’ yields and quality [1, 2]. Manual bioreactor sampling and feeding can be a costly endeavor, both in terms of labor costs as well as increased risk for contamination each time the sterile boundary is penetrated. In this application note, researchers from Merck KGaA, Darmstadt, Germany integrated the ProCellics™ Raman Analyzer via OPC UA (Open Platform Communications United Architecture) connectivity into DASware® control software to optimize their bioprocess, controlled by a BioFlo® 320 bioreactor control system. DASware control allows the easy integration of third-party devices. OPC UA allows the independent implementation into a process while being safer, more stable, and more flexible than older OPC versions (such as OPC DA – Data Access) [3]. The programming of complex feedback loops as functions of different parameters, and the accurate measurements of the Raman analyzer, resulted in stable glucose concentrations without the need of human interaction. Merck KGaA, Darmstadt, Germany develops and commercializes a Raman analyzer for the biopharmaceutical industry, dedicated to in-situ monitoring of bioprocesses. APPLICATION NOTE I No. 415 I Page 2 Material and Methods Media We used FreeStyle™ CHO-S (Gibco®) cells cultivated in CD-CHO medium (Gibco) with 8 mM glutamine, 1‰ of Anti-Clumping Agent (Gibco) and 0.5 % of Penicillin/Streptomycin. Bioreactor control system and process parameters We performed the CHO cultivation, and process monitoring and control with a BioFlo 320 bioprocess controller with a water jacketed 3 L glass bioreactor. The bioreactor was equipped with a ring sparger and a pitched-blade impeller. The DASware control 5.4.1 software was used to control the experiment. The bioreactor was inoculated with cells at a density of 0.4 x 106 cells/mL, with a starting volume of 2 L. Bioreactor settings to control the process are listed in Table 1. The bioreactor was shielded against external light to make sure that the Raman measurements were not affected by external light. Feeding strategy The culture was fed with 15% v/v EfficientFeed™ B (Gibco) on day zero. Glutamine was added when the concentration dropped below 4 mM. On day three, we started with constant glutamine feeding. For glucose feeding, a control loop was programmed based on the glucose concentration: the pump rate of feed B (containing glucose) was controlled by a normal law (on DASware control 5) based on the glucose concentration read by the ProCellics Raman Analyzer to maintain a glucose concentration of 5 g/L. The communication was integrated via OPC UA. The used function was: Model building for Raman monitoring To perform monitoring with Bio4C® PAT Raman Software, a step of model building is needed to correlate the reference values obtained by the BioProfile® FLEX2™ (Nova Biomedical®) and ProCellics Raman Analyzer (Merck KGaA, Darmstadt, Germany). The spectra were preprocessed on the Bio4C PAT Raman Software (SNV on the water region, Savitzky Golay derivative with 3 points (15 cm-1, polynomial order 2nd and 1st derivative) and spectral selection (350- 1775cm-1 + 2800-300 cm-1) to create a dataset. The reference values were automatically linked to their corresponding spectra. The chemometric models for the monitoring are based on four standard fed-batch cultures (total of 103 pt). A PLS model was computed for each monitored parameter using SIMCA® Software (SARTORIUS STEDIM BIOTECH®). Models for Viable Cell Density (VCD), Total Cell Density (TCD), glucose, glutamic acid, ammonium and lactate were performed. Raman monitoring The ProCellics Raman Analzyer acquired and preprocessed Raman spectra, and calculated the process parameters Table 1: Process parameters and cultivation conditions. Parameter Setpoint Control Temperature 37°C Water jacket pH 7.0 (deadband 0.1) Sparging CO2 or 0.5N NaOH pO2 40% Mix of air and O2 sparging (flow rate max 0.1 vvm) Stirring 80 rpm Direct OPC UA communication > Read the spectra > Calculate glucose concentration > Send the glucose value to DASware® control > Read the glucose concentration sent by ProCellics® > Adjust the pump rate for feed (containing glucose) Fig. 2: Experimental setup: The analyzer communicates directly with the biocontroller to send the glucose concentration read in the bioreactor. Fig. 1: Illustration of the complete system setup with the ProCellics probe inside the bioreactor controlled by DASware control 5. Pump rate = 2000e ( -[Glucose]² ) 5 APPLICATION NOTE I No. 415 I Page 3 0.0 2.0 4.0 6.0 8.0 10.0 12.0 0 1 2 3 4 5 6 7 8 9 10 11 12 13 0 24 48 72 96 120 144 168 192 216 VCD (cells/mL) Glucose & lactate concentration (g/L) Culture time (h) A Glucose - Raman monitoring Glucose - O€ line values Lactate - Raman monitoring Lactate - O€ line values VCD - Raman monitoring VCD - O€ line values 0 1 2 3 4 5 6 7 8 9 10 11 12 13 Glucose & lactate concentration (g/L) 0 24 48 72 96 120 144 168 192 216 Culture time (h) 0.0 2.0 4.0 6.0 8.0 10.0 12.0 VCD (cells/mL) Glucose - O­ line values Lactate - O­ line values VCD - O­ line values B Fig. 3: Cell culture parameters evolution. A: Glucose feedback control loop based on Raman measurement (full lines) in comparison with offline measurements for reference (cross) B: Control: Classical fedbatch culture (offline measurements only) including glucose concentration. Based on the scheduled frequency, measurements were carried out every 30 minutes. Following the previously described loop settings, the pump rate was adjusted according to the measurements every 30 minutes. Third-party sensor integration Connectivity between the ProCellics Raman Analyzer and the DASware control software was implemented with OPC UA. Results and Discussion Until day 4 glucose was consumed by the cells until the minimum set value of 5 g/L was reached. The glucose concentration was precisely maintained at 5 g/L for 3 days by the programmed feedback loop (Figure 3A). In parallel, glucose concentrations have been measured offline with FLEX2 in order to assess Raman monitoring. These measurements confirmed that the Raman analyzer accurately measured the glucose concentration. Feeding was stopped when the maximum vessel volume was reached. The process was stopped, when the glucose concentration in the vessel dropped below 1 g/L. As a control, a classical fed-batch run with manual glucose addition was performed (Figure 3B). The cell growth kinetics and the maximum cell density in the feedback-controlled run was comparable with the classical fed-batch control run. However, the lactate concentration in the feedbackcontrolled run was lower (2 g/L) in comparison to the control run (3 g/L). This is a noteworthy result, since to high lactate concentrations can be toxic for cells. Conclusion DASware control 5 enabled the efficient and easy integration of the ProCellics Raman Analyzer via OPC UA protocol. OPC UA is more secure compared to OPC DA. With this setup, we were able to prove that ProCellics Raman Analyzer is fully ready for process automation. Once set up, the automation of the feedback control loop was complete and reliable. A great level of confidence for the extremely stable glucose concentration, and accurately measured with the Raman analyzer was achieved. This allows to reduce the number of human interactions needed, thus reducing the risk of contamination due to repeated sampling. Less sampling is needed due to the automation, resulting in minimized manual work and reducing the risk of contaminations. Additionally, the risk of batch failures due to a lack of glucose during the night or at weekends is reduced. The feeding is stopped at maximum volume TCD VCD Glucose (g/L) Lactate (g/L) The feedback control loop set the concentration at 5 g/L Fig. 4: Cell culture parameters evolution over the cultures, displayed on Bio4C PAT Raman Software. APPLICATION NOTE I No. 415 I Page 4 Ordering information Description Order no. BioFlo® 320, overlay gas option, 1 TMFC (0.05 – 5 SLPM) 1379502111 Software License, DASware® control 5, for one culture vessel 78600166 Vessel Bundle, for BioFlo® 320, water jacket, magnetic drive, 3 L vessel M1379-0311 Pitched-Blade Impeller Kit, magnetic drive, 3 L M1379-5069 Harvest tube M1287-9483 www.eppendorf.com/bioprocess Bio4C® is a registered trademark and ProCellicsTM is a trademark of Merck KGaA, Darmstadt, Germany. BioProfile® is a registered trademark and FLEX2™ is a trademark of NOVA BIOMEDICAL CORPORATION, USA. Gibco® is a registered trademark and FreestyleTM and Efficient FeedTM are trademarks of Life Technologies Corporation, USA. SIMCA® and SARTORIUS STEDIM BIOTECH® are registered trademarks of Sartorius Stedim Data Analytics AB, SE. Eppendorf® and the Eppendorf Brand Design are registered trademarks of Eppendorf SE, Germany. BioFlo® is a registered trademark of Eppendorf, Inc., USA. DASware® is a registered trademark of the DASGIP Information and Process Technology GmbH, Germany. All rights reserved, including graphics and images. Copyright © 2023 by Eppendorf SE. Eppendorf SE reserves the right to modify its products and services at any time. This application note is subject to change without notice. Although prepared to ensure accuracy, Eppendorf SE assumes no liability for errors, or for any damages resulting from the application or use of this information. Viewing the application note alone cannot as such provide for or replace reading and respecting the current version of the operating manual. AA415-020-03-062020 Your local distributor: www.eppendorf.com/contact Eppendorf SE · Barkhausenweg 1 ∙ 22339 Hamburg · Germany eppendorf@eppendorf.com · www.eppendorf.com Literature [1] Brandon N. Berry et al., “Quick Generation of Raman Spectroscopy Based In-Process Glucose Control to Influence Biopharmaceutical Protein Product Quality during Mammalian Cell Culture,” Biotechnology Progress 32, no. 1 (2016): 224–34, https://doi.org/10.1002/btpr.2205 [2] Inn H. Yuk et al., “Controlling Glycation of Recombinant Antibody in Fed-Batch Cell Cultures,” Biotechnology and Bioengineering 108, no. 11 (2011): 2600–2610, https://doi.org/10.1002/bit.23218 [3] Jürgen Lange, Frank Iwanitz, and Thomas Burke, OPC From Data Access to Unified Architecture, 4th rev. Ed., OPC Foundation - Softing (VDE Verlag GMBH, 2010) How can PAT help improving upstream bioprocessing? Find out, which parameters can process analytical technology (PAT) be used to monitor and which sensor types are used. Discover various application examples in which the use of PAT improved the bioprocess! Visit our website www.eppendorf.link/bioprocess-pat APPLICATION NOTE No. 438 Abstract The yeast Pichia pastoris has become an important and convenient workhorse for genetically engineered protein production in the biotechnology industry. Currently, most P. pastoris promoters used for efficient expression of heterologous proteins are derived from genes in the methanol metabolism pathway. For example, PAOX1, the most widely used promotor, requires the switch to methanol feeding to activate protein production, referred to as methanol induction. Due to the flammable nature of methanol, this causes safety concerns in both academia and industry, especially when dealing with large volumes. We explored the feasibility of a safer protein production alternative using a new P. pastoris strain carrying a strong methanol-independent promoter called pUPP. The pUPP promoter requires glycerol feeding for protein production, thus eliminating methanol induction and its affiliated safety risks. We carried out fed-batch P. pastoris fermentation runs for lipase production in the absence of methanol. This workflow was initiated in an Innova® S44i Biological Shaker for inoculum preparation, and followed with BioFlo® 120 and BioFlo 320 bioprocess control systems for bench scale fermentation. We used 3 L BioBLU® 3f Single-Use Vessels as fermenters. At 27 h post inoculation, the BioFlo 120 controlled run reached a maximum OD600 of 183.8. The BioFlo 320 controlled run reached a final maximum OD600 of 229.8 at 46 h post inoculation. Maximum lipase activity was detected simultaneously with peak optical density, reflecting the positive correlation between yeast growth and protein production. We conclude that this methanol-free P. pastoris fermentation workflow can be most advantageous for Eppendorf customers seeking a safer alternative to the traditional methanol induced P. pastoris protein production. From Shaker to Fermenter: Methanol-free Pichia pastoris Protein Production Workflow Ying Yang, Ma Sha Bioprocess Applications Lab, Eppendorf, Inc., Enfield, CT, USA Contact: bioprocess-experts@eppendorf.com APPLICATION NOTE | No. 438 | Page 2 Introduction The yeast Pichia pastoris has been developed as a highly competitive expression system for heterologous protein production. Compared to mammalian cell culture-based protein production systems such as CHO (Chinese Hamster Ovary) cells, the advantages of eukaryotic P. pastoris expression hosts include: > Expedited growth, resulting in drastically shorter fermentation cycle, > robustness, > low cost of medium, > absence of need for viral clearance, > production of large quantities of the desired biomolecules at much higher concentrations [1, 2]. Currently most P. pastoris promoters used for efficient expression of heterologous proteins are derived from genes that code for enzymes in the methanol metabolism pathway. The most widely used promoter is PAOX1 [2, 3], which is strongly repressed in the presence of common carbon sources like glucose and glycerol. Upon depletion of the common carbon source, the promoter is de-repressed and capable of eliciting its full activity when induced by another carbon source, most typically methanol. However, due to its volatility and flammability, the addition of methanol to the bioprocess system brings significant safety concerns. Therefore, alternative promoters which do not require methanol induction to achieve high protein yield are much in demand. Here we used a P. pastoris strain carrying a strong and constitutive promoter pUPP which can rely on glycerol as the only carbon source, meaning that both biomass growth and protein expression are supported by glycerol consumption, whereas no methanol is needed. The objectives of this study are: (1) to carry out fed-batch P. pastoris fermentation with the novel methanol-free induction technology for protein production, here in this case, lipase expression and secretion; and (2) to demonstrate the feasibility of using the Innova S44i Biological Shaker, the two benchtop bioprocess control systems BioFlo 120 and BioFlo 320, and the BioBLU 3f Single-Use Vessel, for high-density P. pastoris fermentation and protein production workflow. Material and Methods Yeast strain The yeast strain Pichia pastoris Bg10-pJAG-Lip1 used in this study was constructed by BioGrammatics, Inc. This strain contains a lipase expression construct and a constitutive pUPP promoter (also known as PGCW14) which is free of methanol regulation in the production of heterologous protein [4]. Upon receipt, we streaked the P. pastoris cells on a YPD agar plate, incubated at 28 °C for 48 h, and picked a healthy single colony to inoculate a 250 mL shake flask containing 75 mL fresh YPD medium. We transferred the shake flask into the Innova S44i Biological Shaker at 28 °C with 200 rpm agitation. After 48 h, we prepared the glycerol stock in multiple cryogenic vials by adding 600 µL of the actively growing P. pastoris suspension from the shake flask to each of the cryogenic vial containing 400 µL 75 % (v/v) sterile glycerol. Cells were well mixed before being transferred into the freezer at -80 °C (New Brunswick Innova U360, Eppendorf) for future use. Media preparation We used two types of growth media in this study, YPD (Yeast extract-Peptone-Dextrose) and BMGY (Buffered Glycerolcomplex Medium). For YPD medium, both agar and liquid media were prepared. The agar YPD medium was made by dissolving 10 g yeast extract (Fisher BioReagents™, BP1422-500), 20 g peptone (BD Gibco™ Bacto™ Proteose Peptone No. 3, 211693), 20 g glucose (Sigma-Aldrich®, G7021), and 15 g agar (BD Difco™ Plate Count Agar, 247940) per 1 L DI water through boiling, and autoclaved at 121 °C. We poured agar plates to streak the P. pastoris cells after receipt as described above. The liquid YPD medium was prepared by dissolving all the compounds except agar at the same amount per 1 L DI water and autoclaved at 121 °C. The liquid medium was used when preparing the glycerol stock. BMGY broth was prepared for bioreactor fermentation. One litre of BMGY broth was made as follows: > 10 g yeast extract, 20 g peptone and 7.5 mL glycerol (Fisher BioReagents™, BP229-1) were disslved in 800 mL DI water and autoclaving at 121 °C. > Next we prepared 100 mL 1 M potassium phosphate buffer (pH 6.0) by dissolving 2.405 g K2 HPO4 (Fisher Chemical™, P228-500) and 11.73 g KH2 PO4 (Sigma Aldrich, P5655) in 100 mL DI water, and sterilized the broth by passing it through a 0.2 µm membrane. We also prepared 10x YNB (Yeast Nitrogen Base, with ammonium sulfate) solution by dissolving 13.4 g of the YNB powder (Invitrogen™, Q300009) in 100 mL DI water and sterilizing through 0.2 µm membrane filtration into the cooled broth to make a total volume of 1 L. APPLICATION NOTE | No. 438 | Page 3 The fed-batch fermentation feeding medium was a 50 % (v/v) glycerol solution, sterilized by autoclaving in the feeding bottle and aseptically connected to the liquid addition port on the head plate of the bioreactor before the run. Inoculum preparation For each procedure, we removed one P. pastoris cryogenic vial from the -80 °C freezer and thawed it under ambient temperature. We prepared two 500 mL Erlenmeyer flasks, each containing 150 mL fresh BMGY medium, and inoculated 500 µL of the P. pastoris glycerol stock from the vial into each of the two flasks. A third control Erlenmeyer flask contained 150 mL fresh BMGY medium without inoculation. We transferred the three flasks into the Innova S44i Biological Shaker for the inoculum to grow at 28 °C with 200 rpm agitation. After 48 h, the broth in the two Erlenmeyer flasks which had been inoculated turned turbid, indicating active growth of P. pastoris. The medium in the control flask should stay clear, demonstrating sterility of the medium and aseptic manipulation. After 48 h shaking culture, we picked one of the two flasks containing 150 mL actively growing P. pastoris suspension and measured its optical density at 600 nm with the fresh BMGY medium as blank using an Eppendorf BioSpectrometer®. The OD600 of this shake flask culture was 30.9. We then carefully poured the 150 mL suspension into two 500 mL sterile bottles. The 75 mL inoculum was pumped at maximum speed into each vessel. The schematic flowchart for glycerol stock preparation and bioreactor fermentation is illustrated in Fig. 1. BioBLU® 3f Single-Use Bioreactor and vessel setup In this study, we performed all fed-batch fermentations in BioBLU 3f Single-Use Bioreactors. The vessel has rigid walls with a working volume range of 1.25 – 3.75 L and allows a maximum agitation of 1,200 rpm. It is equipped with a macrosparger and three Rushton-type impellers, specifically designed for robust microbial applications. All fed-batch fermentations started with an early-stage batch run at 1.5 L working volume in BMGY broth. Towards the end of the fed-batch, the final working volume was around 3 L. We added 0.9 mL Antifoam 204 (Sigma-Aldrich, USA) to the BMGY broth when filling the BioBLU 3f vessel before autoclaving to reach a final antifoam concentration of 0.03 % (v/v). We used a pH/Redox ISM® sensor for pH monitoring and an analog polarographic dissolved oxygen (DO) sensor for DO measurements (Mettler Toledo®, Switzerland). Both sensors are 12 mm in diameter with 225 mm insertion depth. The two sensors were installed on the head plate of the BioBLU 3f vessel through the two Pg 13.5 ports. A stainlesssteel cooling finger was installed through a compression fitting taking another Pg 13.5 port. We extended three liquid addition ports appropriately for connection with the external bottles. Fig. 2 illustrates the BioBLU 3f Single-Use Vessel as it appears before autoclave sterilization. We manually applied the Ni-plated open jaw Hoffman tubing clamp (Eppendorf order No. P0160-4830) to each of the head plate tubing which connects to a submerged port into the medium. These ports include gas sparger, submerged liquid addition line, harvest port, and sampling port. We tightened these metal clamps to prevent the medium from refluxing into the tubing, filters, and possibly ejecting during high-temperature high-pressure autoclaving. Additionally, to ensure effective venting during autoclaving, the overlay gas port on the BioBLU 3f head plate should be left open, as marked by a “Do not clamp” sign preattached to this specific port. We autoclaved the medium-filled vessel and let it cool to 28 °C. We then added sterile 1 M potassium phosphate buffer and 10x YNB solution to the vessel to complete the BMGY broth preparation as described earlier. Upon completion of DO sensor calibration, the inoculation bottle, the feeding bottle, and the base bottle were aseptically connected before inoculation to the liquid addition ports on the vessel head plate through a SCD®-II Sterile Tubing Welder (Terumo BCT, USA). The detailed setup of the vessel can be found in a previously published application note [5]. Fig. 1: Schematic flowchart of the Pichia pastoris fermentation in this study. A. Glycerol stock preparation. B. Bioreactor fermentation. Created with biorender.com APPLICATION NOTE | No. 438 | Page 4 Sensor calibration We followed the same sensor calibration protocols for both BioFlo 120 and BioFlo 320 bioreactors, calibrating the pH sensor outside of the vessel before sterilization. We used the 2-point calibration method by setting ZERO using buffer at pH = 7 and setting SPAN using buffer at pH = 4. We calibrated the pre-polarized DO sensor after autoclaving with the sterile BMGY broth in the vessel. We recommend calibration of the DO sensor under the same condition as during the actual fermentation procedure. Therefore, we checked to make sure the pH was at 6.0, set the agitation at maximum 1,200 rpm, and set the temperature at 28 °C. A 2-point calibration method was also applied. We sparged pure nitrogen at 1 VVM, here in this case 1.5 SLPM, until the DO value stabilized to set ZERO at 0 %; then switched the gas supply to air under the same flow rate, waited till the DO value stabilized again, to set SPAN at 100 %. Pump calibration Prior to the run, the pumps were calibrated on both control systems. The same tubing applied to the peristaltic pump head for liquid addition during fermentation should be used for the pump calibration. It is recommended to add a section of PharMed® tubing (Saint-Gobain®, France) between silicone tubing connections and fit it to the peristaltic pumps for better chemical compatibility especially for base addition. Pump calibration was performed by pumping DI water into a fully filled section of tubing for a set period of time (3 min for both control systems) and tracking the water volume collected in a graduated cylinder at the end of tubing. Then the maximum pump speed specific to the tubing used can be recorded in the system. Pump calibration is critical here for the addition of feeding medium and base during fermentation. Process parameter setup from shaker to fermenters The bench scale P. pastoris fermentations were maintained at 28 °C, pH 6.0, and 30 % DO. Most of the process parameters retained for the two bioprocess control systems (see Table 1). To show the entire workflow, Table 1 also includes the parameter setup of the early inoculum preparation. The key equipment used in this workflow is illustrated in Fig. 3. For the BioFlo 120, heating was controlled by an external heat blanket. The DO cascade was designed to maintain the DO at 30 % by first accelerating the agitation from 300 to 1,200 rpm, then increasing the air sparging rate from 0.8 to Fig. 2: Preparation of the BioBLU 3f Single-Use Bioreactor before autoclave sterilization. A and B, side and top view of the vessel and its head plate; C, D, and E, manually tightened tubing with Ni-plated open jaw Hoffman tubing clamps for gas sparger, submerged liquid addition line, harvest port, and sampling port. a b c d e Table 1. Key process parameters applied to Pichia pastoris fermentation and protein production workflow. Parameter Configuration Inoculum preparation Shaker Innova S44i Biological Shaker Temperature 28 °C Agitation 200 rpm Flask 500 mL Erlenmeyer flasks Volume 150 mL in each Erlenmeyer flask Duration 48 h Bench scale fermentation Controller BioFlo 120 and BioFlo 320 Vessel BioBLU 3f Inoculation density 5 % (v/v), 75 mL inoculum to an initial 1.5 L working volume Dissolved oxygen (DO) 30 %, maintained by DO cascade Agitation Magnetic drive, maximum 1,200 rpm, controlled by DO cascade Gassing Automatic gas flow and mix, controlled by DO cascade Temperature 28 °C, cooling controlled by a single stainless-steel cooling finger pH 6.0 ± 0.1, controlled by the addition of 30 % (v/v) sterile ammonium hydroxide solution Impeller Three Rushton impellers Sparger Macrosparger Feeding Manually triggered by the DO spike, then at a constant feeding rate of 0.4 mL/min of 50 % (v/v) glycerol solution for the rest of fed-batch fermentation APPLICATION NOTE | No. 438 | Page 5 3.0 SLPM, and finally enriching oxygen in the sparged gas stream from 0 to 100 %. These three steps corresponded to the DO output of 0-50 %, 50-65 %, and 65-100 %, respectively, which are fixed in the BioFlo 120. Based on our preliminary studies, to better support yeast growth, we found the importance of keeping a minimum air sparging rate at 0.8 SLPM throughout the run rather than starting from zero air flow. The detailed DO cascade setup in the BioFlo 120 is shown in Fig. 4. For the BioFlo 320, both cooling and heating were controlled by running water through the stainless-steel cooling finger. We set the DO output between 0 % and 100 %, and built a customized DO cascade the same as it was for BioFlo 120 (Fig. 5). For a more direct comparison, Table 2 lists some configuration differences between BioFlo 120 and BioFlo 320 in this P. pastoris fermentation. Besides the aforementioned heating and DO cascades, the most distinctive differences are the gas sparging and pumps. For BioFlo 120, the sparge gas module has 1 TMFC (thermal mass flow controller) which has 4 solenoid valves but only allows one gas to flow through at a given time point without pre-mixing. Therefore, towards the later stage of aerobic P. pastoris fermentation when oxygen enrichment takes place, both air and oxygen are called, but only one gas is sparged to the bioreactor at a given time point depending on the real-time DO output. The BioFlo 320 is equipped with 4 TMFCs. Different kind of gases, air and oxygen in this case, can be called simultaneously at different flow rates and mixed together, thus allowing a more comprehensive and precise control of gas sparging during the process. Furthermore, compared to the three fixed speed pumps on BioFlo 120, BioFlo 320 has three variable speed pumps and a fourth one with much more powerful performance, greatly contributing to the flexibility and efficiency of the bioprocess. An obvious DO spike indicated the depletion of the carbon source, glycerol in this case, in the batch stage of P. pastoris fermentation. When glycerol depletion occurred, the Fig. 3: Key equipment used in this Pichia pastoris fermentation and protein production workflow. Top: Innova S44i Biological Shaker; bottom left: BioFlo 120 bioprocess control station; bottom right: BioFlo 320 bioprocess control station). Fig. 4. DO cascade setup in BioFlo 120. Fig. 5. DO cascade setup in BioFlo 320. APPLICATION NOTE | No. 438 | Page 6 metabolic rate of P. pastoris cells significantly slowed down including the consumption of oxygen. As a result, a DO spike took place. Here the DO spike triggered control was achieved by manual observation and handling with experience from previous runs based on EFT (elapsed fermentation time). We manually turned on the feeding pump at a constant pump rate of 0.4 mL/min right after we detected the DO spike on the trend page collected by both control systems. Biomass formation – optical density measurement Upon completion of DO calibration and right before inoculation, we took a sample of 30 mL fresh BMGY broth from one vessel, and used 1 mL of this medium to set the blank for optical density measurement at 600 nm using an Eppendorf BioSpectrometer. The remaining volume was saved as the diluent for the yeast suspension during the run. We took suspension sample at 11 time points, 0, 3, 6, 21, 24, 27, 29, 46, 48, 51, and 54 h after inoculation, for optical density measurement. Protein production – lipase activity assay The Lipase Activity Assay Kit (colorimetric) used in this study was purchased from Abcam® (ab102524). Since lipase was secreted from the P. pastoris cells, we took one extra mL suspension at the last 7 sampling time points for OD measurements, pelleted the yeast cells down by centrifugation in a MiniSpin® plus microcentrifuge at 14,000 rpm for 90 s, and collected the supernatants for lipase activity colorimetric assay. The basis of this assay is lipase hydrolyzation of a triglyceride substrate to form glycerol which can be quantified enzymatically by monitoring a linked colorimetric change in the OxiRed probe absorbance at 570 nm. We followed the assay protocol to first load a series of standards, samples, background control samples, and positive controls into a 96-well plate, then adding the reaction mix and background reaction mix respectively to the designated wells. The OD570 of the plate was measured by EpochTM Microplate Spectrophotometer (BioTek Instruments) in kinetic mode for 60 min. The standard curve was drawn and the lipase activity of each supernatant sample was calculated accordingly. The detailed protocol is available when purchasing the assay kit or accessed on the Abcam website [6]. Results We ran the Pichia pastoris fed-batch fermentation two times at 28 °C, pH 6.0, and a DO level of 30 % in BioBLU 3f bioreactors, one controlled by a BioFlo 120, and the other by a BioFlo 320 bioprocess controller. We maintained DO by applying a customized DO cascade and initiated feeding right after observing the DO spike. Throughout the fermentation, we took intermittent samples for optical density measurements and lipase activity assays to evaluate the yeast growth and secreted protein production. Bioprocess trends in the BioFlo 120 and BioFlo 320 bioprocess control systems As described previously, a DO spike during fermentation indicates the depletion of the carbon source in the broth and is the signal for initiating feeding. We observed a significant DO spike at 9.25 h in both runs, indicating a synchronized growth and metabolism pattern of P. pastoris in the two vessels. Relative to a DO set point at 30 %, the peak of the DO spike was 35 % and 50 % in BioFlo 120 and the BioFlo 320, respectively. Right before the appearance of DO spike, agitation was ca. 800 rpm and still ramping up. The DO spike was accompanied by a sharp drop of agitation, indicating the largely reduced demand of oxygen since the metabolism of P. pastoris slowed down. Immediately after feeding was initiated, yeast growth quickly resumed, which caused the DO concentration to decrease first and soon recover with agitation continuing its upward trend. For both control systems, the agitation reached its maximum at 1,200 rpm at t = 11.5 h and maintained at this speed for the rest of the fermentation. The air sparging rate ramped Table 2. Differences in bioprocess control between BioFlo 120 and BioFlo 320 in this study. Parameter BioFlo 120 BioFlo 320 Heating An external heating blanket Heated water circulating through the stainlesssteel cooling finger Gas sparging 1 TMFC (0.04-20 SLPM) 4 TMFCs (0.04-20 SLPM) DO cascade Fixed DO output range for a series of defined parameters: agitation, air, and oxygen Can be completely customized, the DO output ranges, and the associated parameters are all self-defined Pumps Three front-mounted pumps (type 114DV) which run at fixed speed with 0-100 % duty cycle Four pumps which can run at variable speeds, the top three are type 114DV pumps, and the fourth has a larger capacity (type 314D) and runs up to 4x faster Inoculation Through one of the three same pumps Through the fourth pump which leads to a very fast inoculation process APPLICATION NOTE | No. 438 | Page 7 up from 0.8 to 3.0 SLPM in the next 3 hours before oxygen enrichment took place at t = 14.5 h. From then on, the trends collected in BioFlo 120 and 320 started to show some difference. In the BioFlo 120 controlled fermenter, oxygen enrichment gradually increased from 0 to 10 % in the sparging gas for the next 7 hours till t = 21.5 h and then started to decline back to 0 % at t = 36 h. Therefore, oxygen enrichment lasted for a total of 21.5 h. After that, the air flow rate started to decrease from 3 to 0.9 SLPM from t = 36 h towards the end of the run at t = 54 h. In the BioFlo 320 controlled run however, oxygen input was only called intermittently at a flow rate of 0.1 SLPM corresponding to 3.3 % in the 3 SLPM sparging gas stream for 1.5 h until t = 16 h, then no oxygen was needed for the rest of the run. The air flow rate started to drop from 3 to 1.6 SLPM during the next 2.5 h till t = 18.5 h and then slowly recovered to 2.4 SLPM at t = 29 h. Beyond that, the air flow rate declined again and was at 1.8 SLPM when we ended the fermentation at t = 54 h. Yeast growth and lipase activity Based on the optical density measurements, growth curves are drawn for both fermentations (Fig. 6). According to the growth curve, P. pastoris grew faster during the exponential growth phase under BioFlo 120 with maximum OD600 of 183.8 at t = 27 h. After that, the stationary phase and death phase were observed in the BioFlo 120 culture and OD600 started to decline till the end of fermentation. However, the growth pattern of the BioFlo 320 culture was different. Although its specific growth rate was lower than the BioFlo 120 counterpart during the early exponential growth, yeast biomass continued to accumulate till t = 46 h with the maximum OD600 at 229.8 in the BioFlo 320 culture before entering the stationary phase. The growth curve correlates well with the culture’s realtime oxygen demand as described earlier. In the first half of the fermentation, after air flow reached its maximum of 3 SLPM at t = 14.5 h, up to 10 % oxygen enrichment was observed for the BioFlo 120 culture and the enrichment lasted for 21.5 h. The early high demand of oxygen supported robust exponential P. pastoris growth controlled by BioFlo 120. However, for BioFlo 320 culture, oxygen enrichment was minimal, but air sparging rate maintained at a relatively high level despite some fluctuations throughout the entire fermentation, which resulted in a long and steady growth of P. pastoris till t = 46 h. For lipase activity, we drew developed a linear standard curve based on the assay protocol to correlate the concentration of glycerol loaded in each well with the OD570 reading associated with the OxiRed probe at the end of 60 min incubation [6]. The equation of this linear standard curve is OD570 = 0.1514 x glycerol concentration (nmol) with a R-square of 0.9937. Therefore, the lipase activity we can be calculated the lipase activity accordingly from the colorimetric readings collected from each well, indicating the glycerol released from triglyceride hydrolysis. We found the maximum lipase activity was 11.5 nmol/ (min·mL) at t = 27 h for BioFlo 120 and 22.5 nmol/(min·mL) at t = 46 h for BioFlo 320. We notice that for both control systems, maximum lipase activity was detected simultaneously with the peak of P. pastoris optical density in each vessel. Since the pUPP is a constitutive promoter, heterologous protein expression and secretion take place at the same time with P. pastoris growth. Therefore, it is not surprising to see Fig. 6. Growth curves of Pichia pastoris and the activities of the secreted lipase in BioBLU 3f Single-Use Vessel controlled by BioFlo 120 (A) and BioFlo 320 (B) bioprocess control systems. 0 5 10 15 20 25 30 0 50 100 150 200 250 0 10 20 30 40 50 60 Lipase activity (nmol/(min·mL)) OD600 Time (h) OD600 Lipase activity 0 5 10 15 20 25 30 0 50 100 150 200 250 0 10 20 30 40 50 60 Lipase activity (nmol/(min·mL)) OD600 Time (h) OD600 Lipase activity a b APPLICATION NOTE | No. 438 | Page 8 that lipase activity and yeast biomass are generally positively correlated, which provides us an easier approach to high protein yield through simply boosting yeast growth. This is very different from growing P. pastoris which has the widely used adopted methanol-induced promoter like such as the alcohol oxidase I promoter PAOX1 for protein production. As a host with such a methanol regulated promoter, P. pastoris needs to go through separate stages for biomass accumulation first and then heterologous protein production by methanol induction, a strategy which poses more challenges in experimental design. Conclusion This study successfully demonstrates a workflow of methanol-free induction for heterologous protein production in P. pastoris from shake flask culture to bioreactor fermentation. The Innova S44i Biological Shaker, the BioFlo 120 and the BioFlo 320 control systems, together with the BioBLU 3f Single-Use Vessels, are all capable of supporting such an innovative and convenient fermentation bioprocess. With a strong and constitutive promoter independent of methanol regulation, the experimental design for such fedbatch P. pastoris fermentation is safer, simpler, and shorter than the traditional PAOX1 induced protein production. P. pastoris reached maximum OD600 of 183.8 at t = 27 h under BioFlo 120, and maximum OD600 at 229.8 at t = 46 h under BioFlo 320. Maximum lipase activity was detected simultaneously with peak optical density, indicating a positive correlation between yeast growth and protein production. This detailed example of methanol-free induction in P. pastoris fermentation can serve as a useful reference for customers seeking a safer alternative to the traditional methanol induced bioprocess. We assert that the methanol-free P. pastoris strain with robust and safe expression system can grow into a key competitor to traditional protein production technologies dominated by the use of methanol induced strains. Literature [1] Pichia pastoris Revisited. Genetic Engineering and Biotechnology News, Vol 34(11). 2014 [2] Ahmad M, Hirz M, Pichler H, Schwab H, Protein expression in Pichia pastoris: recent achievements and perspectives for heterologous protein production. Applied Microbiology and Biotechnology 98(12), 5301-5317. 2014 [3] Gonçalves AM, Pedro AQ, Maia C, Sousa F, Queiroz JA, Passarinha LA. Pichia pastoris: A recombinant microfactory for antibodies and human membrane proteins. Journal of Microbiology and Biotechnology 23(5), 587-601. 2013 [4] Liang S, Zou C, Lin Y, Zhang X, Ye Y. Identification and characterization of PGCW14: a novel strong constitutive promoter of Pichia pastoris. Biotechnology Letters (35), 1865-1871. 2013 [5] Yang Y, Sha M. A beginner’s guide to bioprocess modes – batch, fed-batch, and continuous fermentation. Eppendorf Application Note 408. 2019 [6] ab102524 Lipase Activity Assay Kit (Colorimetric) protocol booklet. Available online at https://www.abcam.com/lipase-assay-kit-colorimetric-ab102524.html APPLICATION NOTE | No. 438 | Page 9 Your local distributor: www.eppendorf.com/contact Eppendorf AG · Barkhausenweg 1 · 22339 · Hamburg · Germany eppendorf@eppendorf.com · www.eppendorf.com www.eppendorf.com/bioprocess Abcam® is a registered trademark of Abcam PLC, UK. Sigma-Aldrich® is a registered trademark of Sigma-Aldrich Co. LLC, USA. ISM® and Mettler-Toledo® are registered trademarks of Mettler-Toledo GmbH, Switzerland. SCD® is a registered trademar of Terumo BCT, Inc, USA. PharMed® and Saint-Gobain® are registered trademarks of Saint-Gobain Performance Plastics Inc., USA. DifcoTM Laboratories Incorporated , USA. Invitrogen™ is a trademark of Life Technologies Corporation, USA. Gibco™ and Bacto™ are trademarks of Beckton, Dickinson and Company, USA. BioReagents™ and Fisher Chemical™ are trademarks of Fisher Scientific Company L.L.C., USA. EpochTM is a trademark of BioTek Instruments Inc., USA. Eppendorf®, the Eppendorf Brand Design, BioBLU®, MiniSpin®, and Eppendorf BioSpectrometer® are registered trademarks of Eppendorf AG, Germany. BioFlo®, Excella®, and Innova® are registered trademarks of Eppendorf, Inc., USA. New Brunswick™ is a trademark of Eppendorf AG, Germany. All rights reserved, including graphics and images. Copyright © 2021 by Eppendorf AG. Ordering information Description Order no. Innova® S44i, incubated, 120 V, orbit diameter 2.5 cm (1 in) S44I200005 Innova® U360, 360 L, ULT freezer, 115 V/60 Hz U9425-0000 New Brunswick™ Excella® E24, orbit diameter 1.9 cm (3/4 in), 120 V/60 Hz M1352-0000 Eppendorf BioSpectrometer® kinetic, 230 V/50-60 Hz 6136 000.002 BioFlo® 320, all configured units include the same base control station Base control station 1379963011 BioFlo® 120 Advanced control station bundle, TMFC 20 SLPM B120ACS000 BioBLU® 3f Single-Use Bioreactor, fermentation, macrosparger, 3 Rushton-type impellers, autoclavable 1386000900 Bioprocess system Accessories Agitation motor for BioBLU® 3f 1386080000 Water manifold kit M1386-9909 Cooling finger for BioBLU® 3f, stainless steel M1386-0555 Exhaust condenser kit M1386-9905 Exhaust condenser bracket, BioBLU® 3f 1386930400 Heat blanket for BioBLU® 3c/5c/5p/3f M1379-8116 Heat blanket adaptor for BioBLU® (to be used with BioFlo® 120) 1386811900 pH/Redox sensor, Mettler Toledo®, InPro 3253i, ISM®, 225 mm P0720-6657 DO sensor, Mettler Toledo®, InPro 6830, angled T-82 connector, 220 mm P0720-6282 Ni-plated open jaw Hoffman tubing clamp P0160-4830 Centrifuge MiniSpin® plus, non-refrigerated, with Rotor F-45-12-11 5453 000 015 APPLICATION NOTE No. 447 Abstract Mammalian cells cultivated in vitro represent one of the most important manufacturing platforms for vaccine and gene therapy developers. Especially, human embryonic kidney 293 (HEK293) cells are an attractive and reliable host for numerous biotherapeutic platforms. HEK293 cells have a wide variety of advantages including low-maintenance, rapid proliferation, and convenient application to both, transient and stable expression. Furthermore, they are easy to transfect and can produce large amounts of recombinant proteins and virus particles. However, a major limitation of the cell line is its tendency to clump when converted to suspension format and therefore has been limited to adherent cell culture. To achieve large scale protein production, a new suspension-adapted HEK293 cell line, Expi293F™, was developed by Thermo Fisher. The new HEK293 cell line appears to be a robust suspension cell line capable of achieving greater per cell productivity in high density culture without clumping. In this study, we evaluated the cell line using bioreactor batch culture. We used a BioFlo® 320 bioprocess control system and a BioBLU® 3c Single-Use Bioreactor to carry out Expi293F batch culture in 2 L scale. In addition we monitored and analyzed the metabolites as well as cell density and viability during 11 days of culture. HEK293 Suspension Cell Culture Using the BioFlo® 320 Bioprocess Controller with BioBLU® 3c Single-Use Bioreactors Jorge L. Escobar Ivirico and Ma Sha Eppendorf, Inc., Enfield, CT, USA Contact: bioprocess-experts@eppendorf.com Interested in more bioprocess information? Our quarterly Bioprocess Spotlight newsletter keeps you up to date about educational material, events, and product news related to your cell culture bioprocess workflow. Subscribe here: www.eppendorf.group/bioprocess-spotlight You can conveniently unsubscribe from the newsletter at any time. APPLICATION NOTE | No. 447 | Page 2 Introduction Developing innovative preventive solutions through new vaccine technologies or gene therapy platforms that respond to new diseases is an important challenge in present day biotechnology. The gene therapy market is projected to reach more than $ 3 billion by 2023 and the global vaccine market is expected to reach $46 billion by 2022, pushing them to the apex of the biotechnology food chain [1-3]. To produce high-quality biotherapeutics, process development involves several demanding components including cost, cell line development, small scale exploration, effective scaling, and optimization of upstream/downstream processes. The selection of the host cell is a key factor based on its capabilities and properties, including its ability to grow in suspension or adhere to a substrate. HEK293 is one of the most versatile mammalian cell lines with a wide range of applications including expression of recombinant proteins, antibodies and viruses. The HEK293 cell line was immortalized in 1973 by the integration of a ~4.3 kbp adenoviral 5 (Ad5) genome fragment containing the E1A and E1B genes, located on chromosome 19 [4,5]. E1A and E1B are essential helper factors for adeno-associated virus (AAV) manufacture, making these cells a popular host platform for AAV particles production [6]. Our goal in this project was to evaluate the suspension culture of Expi293F cell line using Eppendorf bioprocess equipment and assess its suitability in virus production. HEK293 in gene therapy Gene therapy involves the transfer of functional genes into cells to replace absent genes or correct defective ones. In a typical protocol, the cells are extracted from the donor and genetically modified by introducing a new or modified gene to inactivate or to replace a disease-causing gene. These modified cells are then reimplanted in the subject (ex vivo strategy). In addition, a well-established cell culture platform, such as HEK293, can be used to produce viral or non-viral delivery vehicles to introduce the gene of interest (GOI). A well-established method (transient transfection) is frequently employed to produce different vectors using adherent human HEK293 cells cultivated in T-flasks or bioreactors. Prominent examples include lentivirus [7, 8], adenovirus [9,10], non-viral vectors [11], and AAV [12,13]. AAV represents one of the leading platforms for gene therapy due to its ability to provide in vivo long-term gene expression. Nearly 200 AAV clinical trials and biotherapeutic protocols are in different stages of FDA review, in which the transient transfection of adherent HEK293 cells has been the predominant platform [14,15]. We believe that the Expi293F cell line is a significant improvement over traditional HEK293 cell lines due to its robust growth under suspension culture conditions as well as its property of stable expression in addition to transient expression. HEK293 in vaccine production During the last century, vaccines saved billions of lives throughout the world. Vaccine technology has distinguished itself as the most important development in the history of medicine. This unparalleled success has driven researchers to explore new and more efficient platforms to meet the constantly expanding demands of the industry. Conventional vaccines usually contain whole weakened or inactivated viruses or protein subunits made by the pathogen to trigger an immune response. The cell-based vaccine platform is a well-established technology [16], offering several notable advantages: > cell lines are well characterized and may be easily stored for future applications, > their use avoids dependence on embryonated chicken eggs (ECE), whose quality is highly and unpredictably variable, despite their being the most common method used to develop vaccines, > some viruses grow better in cells reducing the time to achieve high growth profiles [17], > compared to ECE, viruses propagated in mammalian cells have shown an antigenic profile similar or identical to that of the field virus [18], and > scalability is superior to the ECE production platform [19]. Although numerous mammalian cell lines have been evaluated for vaccine production [20-24], and performance in gene therapy protocols, the HEK293 cell line is one of the most widely used cell platforms for these demands. Specifically, high yield adenoviral vectors (~4 x 1015 viral particles) have been obtained in stirred-tank bioreactor systems using microcarriers [25,26]. Adherent HEK293 cells are easy to cultivate at laboratory scale, and require less expert bioengineering know-how, but when biotherapeutics production increases, suspension cell lines offer advantages in terms of scalability and robustness, using established stirred-tank bioreactor platforms. Eppendorf bioprocess systems, including BioFlo 320, are not medical devices and cannot be directly used for Gene Therapy and Vaccine production without special approval process. However, it can be used for effective HEK293 suspension cell culture and virus production research including research in the Gene Therapy and Vaccine production field at R&D level. To this end, APPLICATION NOTE | No. 447 | Page 3 Expi293F cells (a suspension adapted HEK293 cell line) can accelerate the therapeutics development by enabling rapid, high yield and scalable production of proteins, viral antigens and AAV particles. In this study, we used a BioBLU 3c Single-Use Bioreactor for Expi293F cell expansion and a BioFlo 320 as the bioprocess control system (Figure 1). We analyzed the cell growth, the viability as well as the metabolic activity (levels of glucose, ammonia and lactate in the medium). Material and Methods Cell line and medium We cultured the suspension Expi293F cell line (Thermo Fisher Scientific, USA) in Expi293 Expression Medium (Thermo Fisher Scientific, USA) formulated with GlutaMAX-I reagent. Expi293 Expression Medium is a chemically defined, serum and protein-free medium, ready to use without need for additional supplements. Inoculum preparation We rapidly thawed the cryovial containing 1 mL of Expi293F cells (Thermo Fisher Scientific, A14527) at 1 x 107 cells/mL, from a previously prepared cell bank, using a ThawSTAR® CFT2 instrument (MedCision®, USA). Just before the cells were completely thawed we decontaminated the vial wiping it with 70% alcohol before opening it in a laminar flow hood. We transferred the entire content of the cryovial into a 125 mL disposable, sterile and vented shaker flask containing 30 mL (24 % of the total volume) of pre-warmed Expi293 Expression Medium (Thermo Fisher Scientific, A1435101). We cultured the cells in a New BrunswickTM S41i CO2 incubator Shaker (Eppendorf, Germany) at 37 °C, 8 % CO2 and at agitation speed of 125 rpm. We cultured the cells for 4 days after thawing and then determined the cell viability and total viable cells using a Vi-CELL XR cell viability analyzer (Beckman Coulter). We then performed the subsequent passages when the viable cell density reached around 3 x 106 cells/ mL (typically 4 days after shaker flask inoculation) reaching more than 900 x 106 cells in the third passage. During the expansion process we kept the inoculation density, percentage fill of the shake flasks and other parameters constant. Finally, we prepared the inoculum containing 800 x 106 cells in 200 mL of Expi293F Expression Medium. The cell expansion workflow is shown in Figure 2. Fig. 2: Inoculum preparation. Created with: BioRender.com. Cryovial of cells ( 1 × 107 cells) 125 mL shake flask Cell density ( 0.4 × 106 cells) 3x 125 mL shake flask Cell density ( 0.4 × 106 cells) 6x 250 mL shake flask Cell density ( 0.4 × 106 cells) BioBLU 3c (2 L working volume) Inoculation density ( 0.4 × 106 cells/mL) Fig. 1: BioBLU 3c Single-Use Bioreactor (left) and BioFlo 320 bioprocess control system (right). Technical Features www.eppendorf.group/bioflo320 In this study, pH, DO, and temperature were controlled online. Get to know more about the possibilities for bioprocess control with the BioFlo 320. APPLICATION NOTE | No. 447 | Page 4 Bioreactor control We used a BioFlo 320 bioprocess control station to perform two batch cultures using BioBLU 3c Single-Use Bioreactors equipped with a single pitched-blade impeller. The bioreactor unit is equipped with two universal port connectors for pH (port 1) and DO (port 2) sensors, an heat blanket connection providing precise temperature control, agitation control and a gas module that includes 3 TMFC, high-flow sparge drawer with a gas flow range of 0.04 – 20 SLPM. Sensor calibration Prior to the preparation of the BioBLU 3c Single-Use Bioreactors, we connected the gel-filled pH sensor to the BioFlo 320 bioprocess controller. The software automatically detects the connected sensors to support an efficient workflow. We performed the calibration process according to the operation’s manual using buffer solutions of pH 7 and pH 4 as “zero” and “span” respectively. Then, we disconnected the pH sensor and sterilized it in an autoclavable pouch. BioBLU 3c Single-Use Bioreactor preparation and process parameters We equipped the BioBLU 3c with a magnetic drive, the previously sterilized pH sensor, inserted in a spare PG 13.5 port under aseptic conditions in the Biosafety Cabinet, a polarographic DO sensor (Mettler Toledo®), an exhaust condenser, a 3-gas mixing line connected to the gas sparge port, and 3 liquid addition ports (one for inoculation/glucose addition, one for base addition and another for the addition of 0.1 % of antifoam (Pluronic®-F68 surfactant, Life Technologies®, 24040-032). Then, we controlled the temperature using a heat blanket. Finally, we introduced the 1.8 L of Expi293F Expression Medium into each bioreactor and conditioned for at least 24 hours under the parameters and setpoints listed in Table 1. Expi293FTM cells culture on BioBLU 3c Single-Use Bioreactor We inoculated the BioBLU 3c Single-Use Bioreactors with the inoculum described above (see section “Inoculum preparation”) reaching 2 L as working volume with a cell density around 0.4 x 106 cell/mL and more than 95 % of cell viability. We monitored the temperature at 37 °C and controlled the dissolved oxygen (DO) at 40 % using the 3-Gas Auto mode. In addition, we limited the oxygen flow to 0 – 1 SLPM and the air flow to 0.04 – 1 SLPM in the controller setup screen to avoid high gas flow that can cause DO fluctuation and excessive foaming in the beginning stage of the cells culture. In addition to the gas flow limit, we added Pluronic-F68 surfactant as needed. We used a gel-filled pH sensor to control the pH during the cell culture run at 7.0 (deadband = 0.2), using a cascade to CO2 (acid) and 0.45 M sodium bicarbonate (base). We took a sample from the bioreactor daily and measured the pH, the cell viability and density as well as the concentration of various metabolites offline. Cell viability and metabolic activity We collected samples on a daily basis from the BioBLU 3c Single-Use Bioreactors to determine the cell viability, cellular density, and the concentration of metabolites (glucose, ammonia (NH3 ) and lactate), by connecting a sterile 5 ml syringe to the Luer Lock sample port. Then, we discarded 5 mL of dead volume and collected again 3 mL (using a new 5 mL sterile syringe) as a viable sample for analysis. We used 1 mL to measure the metabolite levels employing a Cedex® Bio Analyzer (Roche, USA), 1 mL to measure the cell viability and density using a Vi-Cell® XR Viability Analyzer (Beckman Coulter®, USA) and 1 mL to check the pH offline using an Orion Star A211 pH meter (Thermo Fisher Scientific, USA), which we calibrate daily using standard pH buffers. Results and Discussion To evaluate the Expi293F suspension culture robustness, we performed two bioreactor batch culture runs using BioBLU 3c Single-Use Bioreactors controlled by BioFlo 320 bioprocess controller. We used Expi293 Expression Medium with additional glucose supplementation to extend the growth phase and increase the peak cell density. The inoculum was ready after the cell expansion in the New Brunswick S41i CO2 incubator Shaker at 37 °C and 8 % CO2 and agitation speed of 125 rpm. We then inoculated the BioBLU 3c Single-Use Bioreactor with an initial cell density of 0.4 x 106 cells/mL under a controlled environment (see Table 1). Table 1: Process parameters and setpoints of the first and second experiments. Parameters Setpoints Starting volume 1.8 L Ending volume 2 L Initial agitation 120 rpm (0.4 m/s tip speed) Temperature 37 °C Inoculation density 0.4 x 106 cell/mL Cell culture medium Expi293™ Expression Medium DO Setpoint 40% (P=0.1; I=0.001) pH Setpoint 7.0 (deadband = 0.2), cascade to CO2 (acid) and cascade to 0.45 M sodium bicarbonate (base) Gassing range Air flow: 0.04 SLPM -1 SLPM O2 flow: 0 SLPM -1 SLPM APPLICATION NOTE | No. 447 | Page 5 In addition, we added Pluronic-F68 surfactant (0.1 %) to the medium in the bioreactor to decrease foaming produced by the gas introduced through the sparger. As shown in Figure 3A, we observed a rapid increase of cell growth between days 1 and 9 of culture, reaching a peak in viable cells density at 13 x 106 cells/mL, followed by a decrease in cell density and viability as anticipated. Furthermore, we determined the consumption of glucose and production of lactate and NH3 while at the same time maintaining the concentration of lactate and NH3 below 2 g/L and 2 mmol/L respectively during the whole run (See Figure 3B). We performed bolus glucose supplementation (to maintain target concentration > 2 g/L in both runs) at days 3 and 5 to extend the growth phase. The ammonia concentration gradually increased every day up to 2.2 mmol/L on day 11. We believe the depletion of glucose and other nutrients contributed to the decrease of the cell density, starting from day 9. Overall, cell growth increased around 32-fold. Conclusions Using the BioFlo 320 bioprocess control system and BioBLU 3c Single-Use Bioreactors, we demonstrated the feasibility of applying glucose-enhanced batch culture technique to expand Expi293F cells rapidly up to 13 million cells/mL within 9 days. The efficient and straightforward configuration of the BioFlo 320 allows precise control of the cell culture environment, leading to reliable cell expansion. Although the experiments were conducted as feasibility studies and no optimization of conditions was attempted, we observed vigorous growth of Expi293F in suspension culture at a pace and simplicity close to Chinese hamster ovary (CHO) cells, the accepted industry standard. We believe that the Expi293F cell line has great potential in both vaccine and gene therapy method development when used in conjunction with Eppendorf’s advanced stirred-tank bioreactors. Fig. 3 : Expi293F growth profile in BioBLU 3c Single-Use Bioreactor in Expi293 Expression Medium. A: Expi293F cell density and viability. B: Metabolic profile. 0 20 40 60 80 100 0 4 8 12 16 20 Cell viability (%) Cell Density/mL (x106) Time (days) Cell density Batch 1 Batch 1 Batch 2 Batch 2 Cell viability 1 1 1 987 121110 7 6 6 5 5 4 4 4 3 3 3 2 2 2 0 a 0 0 0 1 98765432 121110 Time (days) Glucose (g/L) Lactate (g/L), NH3 (mmol/L) Glucose Lactate NH3 b APPLICATION NOTE | No. 447 | Page 6 Literature [1] Grand View Research, "Gene Therapy Market Size, Share & Trends Analysis," 2019. [2] Markets and Markets, "Gene Therapy Market by Vectors," 2019. [3] Vaccines Market by Indication, Type, Route of Administration & Geography / Global Vaccines Market Forecasts 2018-2025 / ResearchandMarkets.com/CoherentMarketInsights, 2018. [4] Graham FL, Smileyt J, Russell WC, Nairn R. Characteristics of a Human Cell Line Transformed by DNA from Human Adenovirus Type 5. J. gen. Virol 1977; 36:59–7. [5] Louis N, Evelegh C, Graham FL, 1997. Cloning and Sequencing of the Cellular– Viral Junctions from the Human Adenovirus Type 5 Transformed 293 Cell Line. Virology 1997; 233:423–429. [6] Clément N, Grieger JC. Manufacturing of recombinant adeno-associated viral vectors for clinical trials. Mol. Ther. Methods Clin. Dev. 2016; 3:16002. [7] McCarron A, Donnelley M, McIntyre C, Parsons D. Transient Lentiviral Vector Production in HEK 293T Cells Using the BioFlo® 320 Control Station with a BioBLU® 5p Single-Use Packed-Bed Vessel. Application Note 2019; App-411. [8] Ferreira CB, Sumner RP, Rodriguez-Plata MT, Rasaiyaah J, Milne RS, Thrasher AJ, Qasim W, and Towers GJ. Lentiviral Vector Production Titer Is Not Limited in HEK293T by Induced Intracellular Innate Immunity. Molecular Therapy: Methods & Clinical Development. 2020; 17:209-219. [9] Feng L, Wang Q, Shan C, Yang C, et al. An adenovirus-vectored COVID-19 vaccine confers protection from SARS-COV-2 challenge in rhesus macaques. Nat Commun. 2020; 11:4207. [10] Fedosyuk S, Merritt T, Peralta-Alvarez MP, Morris SJ, et al. Simian adenovirus vector production for early-phase clinical trials: A simple method applicable to multiple serotypes and using entirely disposable product-contact components. Vaccine. 2019; 37:6951–6961. [11] Patil S, Gao YG, Lin X, et al. The Development of Functional Non-Viral Vectors for Gene Delivery. Int J Mol Sci. 2019; 20:5491. [12] Wang D, Tai PWL, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov. 2019; 18:358–378. [13] Li C, Samulski RJ. Engineering adeno-associated virus vectors for gene therapy. Nat Rev Genet. 2020; 21:255–272. [14] Rumachik NG, Malaker SA, Poweleit N, et. Methods Matter: Standard Production Platforms for Recombinant AAV Produce Chemically and Functionally Distinct Vectors. Molecular Therapy: Methods & Clinical Development. 2020; 18:98-118. [15] Grimm D, Kern A, Rittner K, Kleinschmidt JA. Novel tools for production and purification of recombinant adeno-associated virus vectors. Hum. Gene Ther.1998; 9:2745–2760. [16] Le Ru A, Jacob D, Transfiguracion J, Ansorge S, Henry O, Kamen AA. Scalable production of influenza virus in HEK-293 cells for efficient vaccine manufacturing. Vaccine. 2010; 28:3661–3671. [17] Jung EJ, Lee KH, Seong BL. Reverse genetic platform for inactivated and live-attenuated influenza vaccine. Exp Mol Med. 2010; 42:116-21. [18] Donis RO. The Influenza Cell Culture Working Group. Performance characteristics of qualified cell lines for isolation and propagation of influenza viruses for vaccine manufacturing. Vaccine. 2014; 32:6583-90. [19] Hegde NR. Cell culture-based influenza vaccines: A necessary and indispensable investment for the future. Hum Vaccin Immunother. 2015; 11:1223–1234. [20] Rimmelzwaan GF, Baars M, Claas ECJ, Osterhaus ADME. Comparison of RNA hybridization, hemagglutination assay, titration of infectious virus and immunofluorescence as methods for monitoring influenza virus replication in vitro. Journal of Virological Methods. 1998; 74:57–66. [21] Voeten JTM, Brands R, Palache AM, et al. Characterization of high-growth reassortant influenza A viruses generated in MDCK cells cultured in serum-free medium. Vaccine. 1999; 17:1942–1950. [22] Pau MG, Ophorst C, Koldijk MH, Schouten G, Mehtali M, Uytdehaag F. The human cell line PER.C6 provides a new manufacturing system for the production of influenza vaccines. Vaccine. 2001; 19:2716–2721. [23] Kistner O, Barrett PN, Mundt W, Reiter M, Schober-Bendixen S, Dorner F. Development of a mammalian cell (Vero) derived candidate influenza virus vaccine. Vaccine. 1998; 16:960–968. [24] Youil R, Su Q, Toner TJ, et al. Comparative study of influenza virus replication in Vero and MDCK cell lines. Journal of Virological Methods. 2004; 120:23–31. [25] Vemula SV, Mittal SK. Production of adenovirus vectors and their use as a delivery system for influenza vaccines. Expert Opinion on Biological Therapy. 2010; 10:1469–1487. [26] Fuenmayor Garcés J, Gutiérrez Granados S, Gòdia Casablancas F, Cervera Gracia L. Cell line and plasmid influence on the efficiency of HEK293 cells transient transfection for the production of VLP-based vaccines. New Biotechnology. 2016; 33:S32-S33. APPLICATION NOTE | No. 447 | Page 7 Your local distributor: www.eppendorf.com/contact Eppendorf SE · Barkhausenweg 1 · 22339 · Hamburg · Germany eppendorf@eppendorf.com · www.eppendorf.com www.eppendorf.com/bioprocess FlowStar® and MedCision® are registered trademarks of MedCision Inc., USA. Mettler Toledo® is a registerd trademark of Mettler-Toledo AG, Switzerland. Pluronic® and Life Technologies® are registered trademarks of Life Technologies Corp. USA. Cedex® is a registered trademark of Roche Diagnostics GmbH, Germany. VI-Cell® and Beckman Coulter® are registered trademarks of Beckman Coulter, Inc., USA. BioBLU®, Eppendorf® and the Eppendorf Brand Design are registered trademarks of Eppendorf SE, Germany. BioFlo® is a registered trademark of Eppendorf Inc., USA. Expi293F™ and Thermo Scientific™ are trademarks of Thermo Fisher Scientific Inc., USA. All rights reserved, including graphics and images. Copyright © 2022 by Eppendorf SE. Ordering information Description Order no. BioFlo® 320, base control station, no water connection 1379962911 BioFlo® 320, left-handed orientation/four front-mounted peristaltic pumps (3 @ 5 – 25 rpm/1 @ 20 – 100 rpm) 1379963211 BioFlo® 320, sparge gas option, 3 TMFC (0.04 – 20 SLPM) 1379501311 Accessories Touchscreen Monitor Bundle, includes desk mount and 1 meter cable set, for BioFlo® 320 M1379-9906 Bioreactor Single-Use Vessel Bundle, for BioFlo® 320, for BioBLU® 3c/5c M1379-0322 BioBLU® 3c, 1.25 L - 3.75 L, Microsparger, 1x pitched-blade 1386000100 BioBLU® 3c ,1.25 L - 3.75 L, Macrosparger, 1x pitched-blade 1386000300 BioBLU® 3c, 1.25 L - 3.75 L, Microsparger, 2x pitched-blade 1386120000 BioBLU® 3c, 1.25 L - 3.75 L, Macrosparger, 2x pitched-blade 1386121000 Executive Summary Plasmids serve as important tool in genetic engineering, gene therapy research or genetic vaccination. Recombinant plasmid DNA (pDNA) is produced in bacterial cultures, mostly in Escherichia coli (E. coli). Successful pDNA production, reaching high yield and quality, relies on multiple factors, including the type of vector and host strain, the insert, and the methodology chosen for cultivation and downstream purification. Here, we review the distinct steps in the plasmid production workflow and give tips on optimization: from vector and strain selection to cultivation, from shake flasks to fermenters up to downstream processing steps of harvesting and purification. From Strain Selection to Purification – Key Factors for Successful Plasmid Production Introduction Plasmids serve as a tool in genetic engineering, either to clone and amplify DNA fragments, such as genes, express recombinant proteins or to serve as templates e.g. for in vitro mRNA production or transient CRISPR expression in gene therapy research and genetic vaccination [1,2]. Plasmid DNA (pDNA) can be easily genetically manipulated and produced in large amounts in bacteria, typically in E. coli. Furthermore, a variety of ready-to-use solutions allows easy subsequent downstream purification. WHITE PAPER No. 89 Stefan Schlößer¹, Eric Vincent3, Ruchika Sharma3, Samuel Ellis4, Ines Hartmann2 1 Eppendorf SE, Jülich, Germany 2 Eppendorf SE, Hamburg, Germany 3Promega, Madison, WI USA 4Thomson Instrument Company, Oceanside, CA USA Vector selection & design Host selection + transformation Shake culture Fermentation Harvesting Purification Depending on the application, pDNA production ranks from research laboratory scale (microgram range) to large industrial scale (milligram to gram range). The plasmid production includes multiple steps (Figure 1). In the following, we review the factors affecting plasmid production and give tips on optimization. The production of pDNA can be separated in five parts: 1) Plasmid vector selection, page 2–3 2) Host strain selection and transformation, page 3 3) Production in shake flasks and fermenters, page 3–12 4) Culture harvesting, page 12 5) Purification, page 12–13 Figure 1: Plasmid production workflow WHITE PAPER I No. 89 I Page 2 1) Plasmid vector selection Since the availability of the first commercial cloning plasmid vectors in the 1970s, the number of available products has increased substantially. Plasmids are small circular double stranded DNA molecules. The typical size ranks from thousand up to a few thousand base pairs. Minimal required components of plasmids used in the lab include the origin of replication (ori), which enables the independent replication from the host’s chromosome, the multiple cloning site (MCS) or polylinker region with the restriction digestion sites, that allows to insert the gene of interest, and a selection marker to ensure that only bacteria containing the plasmid survive in culture. In expression plasmids, an additional promoter is inserted upstream to the multiple cloning site, which drives the transcription of the gene (Figure 2). Figure 2: Minimal components of a plasmid vector. Important components include the origin of replication (ori), the multiple cloning site with the restriction sites (here shown with the inserted gene of interest) and the selection marker (here an antibiotic resistance gene). In expression plasmids an additional promoter is inserted upstream the multiple cloning site. The following parameters are helpful for choosing the right plasmid for your needs. Replicon and copy number One important factor affecting the yield of plasmids from a given system is the copy number, meaning the expected number of plasmids per host cell. A plasmid’s copy number is determined primarily by the ori and the surrounding regulatory DNA sequences. This area, known as the replicon, controls the replication of plasmid DNA. Plasmids can be grouped into low and high-copy plasmids (Figure 3). For example, plasmids derived from the plasmid pBR322 contain the ColE1 origin of replication from pMB1. This origin of replication is tightly controlled, resulting in only ~25 copies of the plasmid per bacterial cell (low copy number). On the other hand, pUC-derived plasmids contain a mutated version of the ColE1 origin of replication, which results in reduced replication control and ~200–700 plasmid copies per cell (high copy number). Figure 3: High and low copy plasmids. High copy plasmids (left) result in increased plasmid copies per cell, compared to low copy plasmid (right). In most cases, a high-copy plasmid produces greater yields of plasmid DNA and is the preferred choice for gene cloning. Another advantage of the high copy number is higher plasmid stability when random segregation occurs during cell division [3]. However, high-copy plasmids pose a high metabolic burden on the host cell. That may lead to poor culture growth and can favor plasmid instability. Especially for ambitious cloning tasks, like amplifying plasmids with large inserts >8 kb or adenine and thymine rich (‘AT-rich’) sequences, a low copy plasmid may be considered to increase stability [4]. Excursus I: Considerations for protein expression plasmids For most protein expression applications, high-copy plasmids are well suited to produce high amounts of protein. However, when high-level expression results in protein aggregation, a slowed-down synthesis rate can have a positive impact on yields and a low copy plasmid may be considered [5]. Other measures, such as introducing additional backbone features into a proteinexpression plasmid (for example protein-solubility increasing fusion tags), using weaker promotors, or co-expressing multiple protein components with a compatible dual expression plasmid system, are strategies to further improve solubility [6]. Bacteria cell Plasmids WHITE PAPER I No. 89 I Page 3 Insert size Most commonly available plasmids like pUC and pBlueScript can take inserts of up to around 15 kb [6]. However, larger fragments can lead to decreased plasmid copy numbers or problems in plasmid DNA replication and stability [7]. There are special types of high-capacity vectors such as PACs, BACs, cosmids, which are suitable for cloning large DNA fragments [8]. Selection marker Almost all plasmids have a selection marker, usually an antibiotic resistance gene, that ensures that only the cells that have incorporated the plasmid vector can survive in the presence of the specific antibiotic. Depending on the selection marker, the referring antibiotic must be added to the solid or liquid medium to keep the selection pressure and plasmid stability. To avoid degradation, antibiotics should be added to the medium after autoclaving at temperatures lower than 60 °C and always freshly prior use. For some therapeutic plasmid-based products, the presence of antibiotics is restricted or not recommended by regulatory authorities. Kanamycin is the most widely used and preferred selection agent for DNA vaccines, as it does not present a significant allergic risk [9]. Engineering more stable vectors and alternative selection strategies to antibiotics have been investigated [10]. 2) Host strain selection and transformation The choice of the E. coli host strain can have a significant impact on the quantity and quality of the produced pDNA. E. coli K-12 strains such as DH5α, JM109 and the slower growing XL1-Blue are common host strains for gene cloning applications generating high plasmid yields. They contain several gene mutations, including one in the endonuclease A (endA) gene to prevent plasmid degradation and one in the recombinase A (recA) gene to avoid homologous recombination. Constant genetic engineering efforts, such as metabolic engineering strategies targeting individual bacterial enzymes relevant to the cell’s physiology, have been explored with the aim of increasing plasmid production [11,12]. Excursus II: Host strain selection for protein expression Amongst protein expression strains E. coli B-strains, such as BL21 (DE3), are often chosen for high-level production of recombinant proteins. They are deficient in genes encoding certain proteases that can degrade proteins during purification. Several strain modifications for difficult protein expression have been engineered, such as BL21(DE3)-RIL for expression of rare codon-containing genes and BL21-AI for toxic proteins. Two procedures are primarily used to transfer plasmids into bacterial cells: chemical transformation and electroporation. Chemical transformation or heat shock is cost-effective and well established for E. coli but is a more time-consuming and labor-intensive method compared to electroporation. Electroporation requires investment in a specific electroporation device and cuvettes but has been shown to increase transformation efficiency [13] and to be beneficial for larger insert sizes [14]. The transformation is followed by clonal selection on agar plates containing selection markers, such as antibiotics. Transformation efficiency can be calculated as the number of colony-forming units (CFU) per microgram of plasmid DNA used. 3) Production in shake flasks and fermenters Cultivation technique – choosing a shake flask or fermenter The growth environment plays a crucial role in pDNA production. Productivity is usually proportional to the final cell density and the specific productivity (amount of pDNA per unit cell mass). The pDNA yield is commonly indicated in milligrams of pDNA per liter of culture (mg/L). Typically, 0.5–1 g pDNA per kg of wet weight biomass is obtained from using high copy plasmids [15]. Besides existing lab resources, the application and yield demands determine the cultivation method of choice. Tube cultures and small shake flasks are suitable for gene cloning and laboratory scale protein production, where working volumes between 1 mL to 1 L per vessel and yields in the microgram range are usually sufficient. If higher plasmid yields are required, for example for transfection purposes, large-scale recombinant protein production, or if the plasmid is the final product itself in genetic vaccination or gene therapy applications, a controlled fermenter is usually the device of choice, being able to handle higher working volumes. Knowing the bacteria´s growth behavior Growth curve measurements based on optical density (OD600) provide a simple tool to analyze the bacterial growth behavior, for example to compare different strains, analyze the influence of media composition, or determine the optimal point of harvesting. In a typical growth curve, one can differentiate four distinct phases of growth: the lag, exponential (log), stationary, and death phase (Figure 4). WHITE PAPER I No. 89 I Page 4 0.1 for shake flasks (often higher for fermenters). However, the optimal inoculation density is dependent on the growth characteristics of the microorganism and the given growth environment and can be evaluated by performing initial growth curve comparisons. Unlike mammalian cell cultures, long, cost-intensive seed trains are not required, and, in most cases, a one-stage inoculum train is sufficient also in industrial settings [20]. Stationary phase B b t [days] Figure 4: Typical bacteria growth curve (batch culture). In the initial lag phase, the cells are in a metabolically active state, but there is no or only little cell division. The cells accommodate to the new conditions. The subsequent logarithmic (log) phase is the phase of optimal growth with cell numbers increasing in a logarithmic fashion. In the stationary phase the cell growth rate slows upon nutrients depletion or toxic product accumulation and the rate of cell division and death is roughly equal. In the death phase the cell number decreases. In red: The generation or doubling time G of the cells is calculated during the exponential phase of growth. B is the number of bacteria present at the start of the observation, b is the number present after the time t. From the growth curve, one can calculate the generation (=doubling) time (G) in minutes or hours using the following formula: G = t n = t 3.3 log b / B In the formula, B is the number of bacteria present at the start of the observation, b is the number present after the time t, and n is the number of generations. The generation time is calculated during the exponential growth phase (linear growth on a log-scale plot) (Figure 4). Inoculation strategies The most frequently recommended method to inoculate a small liquid culture from a frozen stock, is to start from a single colony grown on a freshly streaked selective agar plate [16,17,18]. This ensures that the culture derives from one clone. However, a recent study comparing direct inoculation from a glycerol stock versus inoculation from a single colony did not identify significant differences between the two methods in terms of growth behavior and final plasmid yields [19]. Shake cultures and fermenter cultures are often inoculated from a liquid pre-culture to reduce the lag-phase (Figure 5). The pre-culture should be harvested within the logarithmic phase. Inoculation with a pre-culture does not only shorten the lag phase, but also allows a better synchronization of cultures by adjusting to a defined start OD600. This is helpful when comparing strains. A typical starting OD600 is around 0 1 2 3 1 2 3 4 5 6 OD600 Time [h] Agarcolony Liquid pre-culture Figure 5: Influence of inoculation method on bacteria growth. Inoculation of 250 mL Erlenmeyer flasks filled with 20 % LB medium with either a single colony from an agar plate or from a liquid pre-culture, starting OD600 ~0.04. (E. coli DH5ɑ, incubation at 37 °C and 250 rpm) The optimal harvesting timepoint The harvesting time point affects both the yield and quality of plasmid DNA. Bacterial cultures grown to insufficient density will yield reduced amounts of pDNA. Overgrown cultures may result in suboptimal yields and excessive chromosomal DNA contamination due to autolysis of bacterial cells after they have reached the stationary phase. Harvesting for plasmid preparation is usually recommended in the late logarithmic or early stationary phase [21]. By this time, the culture possesses high biomass, hence plasmid yield, but not too many dead cells. Growth times for cultures with high copy plasmids under standard conditions are typically between 12-16 hours, and for cultures with low copy plasmids ≥ 20 hours [18]. Due to the higher metabolic burden posed on the bacterial cell by high copy plasmids, the achievable maximum cell density of these cultures is usually lower compared to low copy plasmids, resulting in a shorter incubation period. The optimal cultivation time may vary for several reasons, such as the utilized strain, plasmid, temperature, agitation speed, flask type, media composition, and cultivation mode and should be evaluated based on growth curve analysis and sampling at various time points with subsequent plasmid preparations. WHITE PAPER I No. 89 I Page 5 Optimizing tube culture For small-scale approaches culturing is typically carried out in 15 mL conical tubes. However, due to their small diameter, handling can be difficult. They are also not optimal for efficient aeration due to the small surface-to-volume ratio. A 25 mL conical tube such as the one provided by Eppendorf has the diameter of a 50 mL tube offering the advantage of a larger surface for oxygen transfer but is half the size of a standard 50 mL tube, thus better accessible with a pipette. A comparison between the 25 mL Eppendorf conical tube and a standard 15 mL tube, using the same inoculum amounts of E. coli, identified enhanced cell numbers and plasmid DNA yields in the 25 mL tube [22] (Figure 6). This indicates improved mixing and aeration due to the different shapes. As a rule of thumb, fill volumes in tubes should not exceed 20 % to facilitate adequate mixing. In addition, an increased liquid surface for oxygen supply can be achieved by cultivating the tube at an angle such as 30-45 °, especially when working at standard shaking speeds of around 200 rpm [23]. 800 600 400 200 15 mL culture 25 mL tube 7.5 mL culture 25 mL tube 7.5 mL culture 15 mL tube 0 Plasmid DNA Yield [µg] Figure 6: Influence of tube design and size on plasmid yields. Total yield of low copy plasmid DNA (PBR322) purified from bacterial cultures E. coli DH5α incubated in Eppendorf Conical Tubes 25 mL with snap cap and standard 15 mL conical tubes. Inoculated from one seed stock and grown under similar conditions. Optimizing shake flask culture Shake flasks are the most used cultivation vessels for production of plasmids on laboratory scale. Also, early process development, such as initial strain and media selection, starts typically in shake flasks prior to further process development and expansion in fermenters. Shake flasks are inexpensive, easy to parallelize and simple to use. There are different parameters one can adjust in cultivation to increase biomass and product yields in a shake flask. Although E. coli is a facultative anaerobic microorganism, it grows best in the presence of oxygen [24]. In a shake flask, oxygen supply only occurs passively via the air-liquid interface. Oxygen limitation is one of the most frequent problems in shake flask culture. Different shake flask designs are available to increase the oxygen transfer to the culture (Figure 7). Figure 7: DIfferent shake flasks A) Classic non-baffled and baffled Erlenmeyer flask designs. Baffled flasks have defined cavities in the bottom area B) Ultra Yield® flasks. Flasks with steep vertical walls and six baffles at the flask bottom in 2.5 L size (Thomson). Baffles for example, disrupt the regular swirling liquid flow by the implemented cavities in the bottom area, thus improving the aeration of the culture [25]. Comparisons of different flask designs have shown that baffles can improve cell growth and maximum cell density drastically compared to unbaffled flasks (Figure 8). 0 5 10 15 20 25 30 2 4 6 8 24 OD600 Time post inoculation [h] Ultra Yield® Baed Non-baed Figure 8: Influence of flask design on bacterial growth. Ultra Yield® flasks with the specialized baffled design resulted in the fastest growth and maximum cell density, followed by the standard baffled design and finally the non-baffled design (E. coli DH5α with pUC19 plasmid cultivated in modified TB media in 500 mL flasks with 25 % fill volume, 37 °C and 200 rpm). The disadvantage of this design is a higher risk of foam formation that may lead to cross contamination by wetting the flask closure. However, this problem can be solved by using an antifoam agent. Unreproducible results caused by variances in baffle geometry and size have been described for glass flasks [26]. However, this has not been seen in disposable flasks [27], assuming these effects may be dependent on the manufacturing process in general. a b WHITE PAPER I No. 89 I Page 6 Typical agitation speeds for flask cultures are 200- 250 rpm. An increase in agitation speed supports the oxygen transfer to the liquid and increases both biomass and plasmid yields independently of flask size [28]. An increase from 250 to 350 or 400 rpm resulted in up to almost 2-fold higher biomass [data shown in original app note 449] and a ~30-90 % increase in pDNA yields in 500 mL and 2.5 L Ultra Yield® flasks, respectively [Figure 9A and B]. However, care must be taken, especially when using high agitation speeds with baffled flasks, to prevent cap wetting. 34.8 45.2 0 50 100 pDNA yield [mg/L] 250 rpm 400 rpm A) DH5α transformed with pUC19 64.0 120.3 200 rpm 350 rpm B) JM109 transformed with pGEM-3Z Figure 9: Influence of agitation speed on pDNA yields. An incubation at higher agitation speed resulted in higher pDNA yields for both E. coli strains. A: Cultivation of E. coli DH5α transfected with pUC19 plasmid in 2.5 L Ultra Yield® flasks (20 % fill, 37 °C, modified TB media, harvest 8 h post inoculation). B: E. coli JM109 with pGEM®-3Z plasmid in 500 mL Ultra Yield® flasks (25 % fill, 37 °C, modified TB media, harvest 8 h post inoculation). As a rule of thumb, fill volumes in a shake flask should not exceed 20 % for microbial applications. In case of problems with oxygen supply, one can further reduce the fill volume to 10 or even 5 %. Some baffled designs, such as Thomson Ultra Yield®, may allow also higher fill volumes under certain conditions [28]. Again, when using higher fill volumes in combination with agitation and/or baffles, care must be taken to avoid cap wetting. Flask size can also influence pDNA yields. A smaller flask will give higher maximum oxygen transfer rates compared to larger flasks (when similar conditions, same fill and agitation speed are applied) due to the relative volumetric surface area increase. Oxygen transfer rate (OTR) measurements with 2.5 L and 250 mL Ultra Yield flasks have been described [27]. Hence, when scaling up from small to large flasks, conditions may need optimization, such as agitation speed or fill volume to get similar yields per L. Influence of temperature The standard cultivation temperature for E. coli is 37 °C. However, for some plasmids the copy number can be increased by a temperature up-shift to 42 °C. Temperature sensitivity has been described for high copy number pUC plasmids and explained with the existence of certain mutations [29]. This strategy of induced amplification is used primarily in fermenters. Typically, fermentation starts with a lower temperature at 30°C, at which the plasmid is maintained stably at low levels while biomass is accumulating. When cell density has reached a certain level, the plasmid amplification is induced by shifting the temperature to 42°C and by continued exponential nutrient feeding for a defined time [29]. It has also been reported that plasmid yield and quality can be improved by holding fermentation cells at a reduced temperature post the 42°C induction phase [30]. Excursus III: Influence of temperature on protein expression A cultivation at reduced temperatures may reduce the problem of protein misfolding and aggregation and can be one factor to optimize when working with difficult to express proteins. Influence of media composition The media should support cell growth as well as plasmid amplification and stability while minimizing other cell activities. Other than in mammalian culture, the media costs are relatively low due to the mostly simple formulations. One distinguishes minimal (defined) media and complex (semi-defined) media. In minimal media the exact chemical composition is known, whereas complex media contains components of unknown chemical composition and varying proportions, such as yeast extract or peptones for example. Minimal media allows for highly reproducible processes [31]. Complex media allows for high cell densities but may give more varying results and might interfere in downstream processing in industrial settings [32]. Formulations based on Lysogeny Broth (LB) or Terrific Broth (TB) are frequently used complex media. Bacteria need as minimum set of nutrients to grow: water, a carbon source, a nitrogen source, and some mineral salts. However, typical bacterial complex components are protein hydrolysates like peptone or tryptone, yeast extract, carbohydrates, nitrogen, trace metals, and minerals. Glucose is the most common carbon source added, as it is inexpensive and can be efficiently metabolized by E. coli, leading to good biomass and specific plasmid yields [33]. However, it might favor the undesirable accumulation of acidic by-products, such as acetate, due to metabolic overflows. Acetate accumulation is a major concern in aerobic fermentation known to inhibit cell growth and protein expression [34]. However, the effect of acetate on plasmid DNA production has not been investigated much. One study states that acetate formation can improve pDNA yields when present at low concentrations (3 g/L) [35]. Reduced growth rates have been linked to elevated copy numbers before [36]. Glycerol can be chosen as an alternative or complementary carbon source. It increases specific plasmid WHITE PAPER I No. 89 I Page 7 productivity but may lower cell growth compared to Glucose [36]. The carbon sources of LB media are metabolizable amino acids in the form of tryptone and yeast extract with no additional carbon source added. Beside a higher amount of yeast extract and glycerol, TB media formulations contain an additional carbon source, which may increase pDNA yields compared to classic LB formulations (Figure 10) [27]. LB medium is excellent for routine molecular biology applications, but bacteria growth is limited due to the presence of the small amounts of utilizable carbon sources [37]. 7.1 34.8 0 10 20 30 40 50 LB media Modified TB media pDNA yield [mg/L] Figure 10: Influence of media on pDNA yields. The TB media formulation resulted in higher pDNA yields compared to LB media. Cultivation of E. coli DH5α with pUC19 plasmid in 2.5 L Ultra Yield® flasks in either LB or modified TB media (20 % fill, at 37 °C and 250 rpm). Nitrogen is important to produce proteins and nucleic acids and can be either supplemented from organic sources, such as yeast extract or hydrolysates like tryptone/peptone or from inorganic supplemented nitrate. Beside the availability of nutrients, the pH of the medium affects the culture. The optimal pH range for E. coli is between 6.5 and 7.5, depending on temperature [38]. During growth, the pH is drifting as consequence of production of substrates and metabolic compounds. A decrease of pH occurs for example by acetate accumulation due to metabolic overflow when cultivating with glucose as a carbon source. However, after glucose depletion consumption of acetate by the bacterial cells causes the pH to increase again [39]. Hence, pH control is important, especially when the medium contains additional carbon sources like glucose. Phosphates are commonly used buffer systems. Alternative buffer solutions are zwitterionic buffers like MOPS [40]. An overview of commonly used media with a short description of their properties can be found in online sources of suppliers [41] There are also commercially available ready-to-use media optimized for plasmid production that support high-density growth and high plasmid yields (Table 2) [42]. Table 2: Plasmid+® Media yields higher plasmid concentrations over longer time periods compared to other complex media. Shown are the plasmid yields of Plasmid+ versus commonly used TB medium cultures in 2.5 L Ultra Yield flasks. Plasmid concentrations are given in ng/µL. Yield differences between Plasmid+ and TB medium cultures are given in percent. Experiments were done at 310 rpm and 37 °C (Source: Thomson) Yield [ng/µL] % increase vs TB Incubation time [h] 20 hr 22 hr 24 hr 20 hr 22 hr 24 hr Plasmid+, at 310 rpm 94.9 103.3 142.6 190.2 274.3 354.1 Terrific Broth, at 310 rpm 32.7 27.6 31.4 – – – Traceability and source documentation for media components, such as batch certificates of analysis, are especially important in regulated areas. The trend in the industry towards eliminating the use of any animal-derived components or even completely chemically defined medium to alleviate potential regulatory concerns has been described [43]. Optimizing fermenter culture A bioreactor or fermenter in microbial settings is a vessel that provides a biological growth environment for the (micro)organism of choice and is usually used together with a bioprocess control station and software to monitor, adjust and maintain this environment (Figure 11). Figure 11: Composition of a bioreactor. Components of a stirred tank‐bioreactor (left) and a connected control station (right). a: bioprocess control software b: motor c: pump d: head plate e: sensor f: bioreactor g: bioprocess control station h: impeller a b c d e f g h WHITE PAPER I No. 89 I Page 8 The biggest advantage of bioreactors is a stable and adjustable growth environment due to precise control of various incubation parameters, such as temperature, agitation speed, gas supply and pH. Furthermore, it offers the possibility to scale up the bioprocess which enables reaching increased cell numbers and plasmid yields [44]. However, also bioreactors offer certain challenges that can influence process efficiency. This section aims towards overcoming them and giving insights into the most common considerations for the use of bioreactors. For this, we will look at the example of the stirred-tank bioreactor type which utilizes a motorized impeller to swirl the bacterial cell suspension to support gas exchange, nutrient distribution, and culture homogeneity (Figure 12). The topics of this section include the method or mode of fermentation, considerations concerning culture conditions and equipment, as well as bioprocess scale-up. Fermentation mode When starting fermentation in a bioreactor, one has the choice between three commonly used fermentation modes, namely batch, fed-batch and continuous fermentation. In batch fermentation, inoculation is carried out in a fixed volume without the addition, removal, or exchange of medium or feeding solution. In the process of nutrient consumption, the culture will undergo the typical cellular growth phases: first a lag phase in which the microorganisms adapt to the new environment, then an exponential phase marked by rapid cell division and cell density increase, followed by a stationary phase where growth is halted due to nutrient limitations and lastly a death phase in which the cells start to die [45]. Batch fermentation is easy to set up and bears a low contamination risk. However, the microorganisms experience a constantly changing environment due to the ongoing nutrient depletion and accumulation of potentially toxic by-products within the medium [45]. This might affect the growth of the culture resulting in lower cell density and ultimately decrease plasmid yields. Therefore, fed-batch fermentation is an advancement of the batch mode to overcome its disadvantages. Here, nutrients are replenished during the incubation creating a more stable environment for the cells. Usually, the feeding is initiated during the mid-exponential growth phase (OD600 of 8-12) to prevent nutrient deprivation [46]. Thus, fed-batch not only enables higher cell density and biomass production than batch fermentation, but it also allows for longer cultivation periods and limits by-product accumulation [45, 47]. Consequently, higher plasmid yields were observed for fedbatch over batch fermentation [48, 49]. However, the more complex structure of the fed-batch process also increases the risk of contamination [45]. Thus, care needs to be taken when preparing and adding the required feeding solutions. The last fermentation method we look at, continuous fermentation, is the most complex operation mode. However, once it is ideally adjusted long cultivation of days, weeks and even months are possible. This main advantage is achieved by continuous removal of used medium and cells while fresh medium and nutrients are added simultaneously [45]. The resulting steady state ideally keeps the volume and nutrient concentrations constant while simultaneously removing toxic by-products, thus creating the most stable cell environment yet. Furthermore, the long run times reduce process costs and downtimes compared to batch and fed-batch, for example, due to fewer cleaning procedures [45, 50]. However, the greatest strength of continuous fermentation also causes a lot of its weaknesses. Long-term incubation makes maintaining sterile and stable growth conditions more challenging. As a result, product yields are usually lower than for batch and fed-batch fermentation which increases downstream costs [50]. Therefore, if long-term production of plasmids in a bioreactor is not your main concern, fed-batch fermentation is recommended as the best synthesis in terms of productivity, downtime, and contamination risk. Table 3 summarizes the advantages and disadvantages of each fermentation mode. Batch Fed-Batch Continuous Figure 12: Bioreactor operation modes. Batch cultivation carried out in a fixed volume, fed‐batch with medium and nutrient addition after the initial lag‐phase until the end of the process, and continuous cultivation with constant medium/nutrient replenishment to ensure a stable growth environment. WHITE PAPER I No. 89 I Page 9 MODE PROS CONS Batch > Ease of use > Low contamination risk > Constantly changing environment > By-product accumulation > Low cell density > High downtime for cleaning due to short run-time Fed-batch > Longer cultivation time compared to fermentation and thus less downtime > More stable environment than batch fermentation due to feeding > Higher cell density possible compared to batch fermentation > Consequently, higher plasmid yield than batch fermentation > By-product accumulation limited > More complex structure than batch fermentation > Higher contamination risk than batch fermentation due to feeding Continuous > Longest possible cultivation period (weeks to months) > Ideally a constant environment > Growth rate constant > Possibility to remove by-products and prevent accumulation in the culture > Strongly reduced downtime > Most complex process structure > Highest contamination risk of all operation modes > Compared to fed-batch lower productivity (cell density, plasmid yield) Table 3: Pros and cons of common fermentation modes Culture conditions and equipment Once the fermentation mode is selected, the culture needs to be maintained. Many parameters impact the efficiency of cultivation and most of them can be precisely controlled in a bioreactor. The first and foremost is the optimal growing temperature which is 37°C for most bacteria. As opposed to shake flasks that need to be transferred to an incubator, the temperature of a bioreactor can be set, controlled, and automatically adjusted in place [51]. This is achieved by temperature probes, as well as heating blankets, thermowells, water jackets, and cooling fingers which can provide heat to or remove heat from the medium. Another factor important for bacterial growth and plasmid yield is the pH value. Of course, the optimal value varies from microorganism to microorganism. However, in most cases, Escherichia coli is used for plasmid production. Here, we will look exemplarily at E. coli DH5α, one of the most commonly used strains for plasmid production. Studies in tubes and shake flasks identified its optimal pH range for plasmid production to be between 6.7 and 7.5, depending on the plasmid and the medium type [52, 53]. However, it is worth mentioning that the pH changes over the course of nutrient consumption and growth. Studies showed that the pH usually drops over the course of incubation [53,54]. As pH shifts can impact the resulting plasmid yields, it is desirable to monitor and adjust the pH accordingly, something that is more difficult to achieve in shake flask cultures. Bioreactors on the other hand offer precise and continuous pH measurements through sensors with the option of automatic adjustments by adding either acid or base solutions to the medium [51]. Ensuring efficient mixing and aeration of the culture is also critical for the bioprocess. Therefore, agitation speed and the right impeller type need to be selected for optimal culture growth. Both parameters are usually a tradeoff between sufficient aeration, mixing, and prevention of shear stress on the cells. However, as most bacteria have a relatively low shear sensitivity, a commonly used Rushton or Rushton-like impeller (Figure 13) with high agitation speed can be recommended for bacterial fermentation [55]. Figure 13: Rushton-type impeller used for bacterial bioreactor cultures with low shear-sensitivity. WHITE PAPER I No. 89 I Page 10 If the impeller is not sufficient to aerate the culture efficiently, additional gassing can help. Oxygenation is reached by addition of O2 , while anaerobic conditions can be achieved by gassing with nitrogen or other anaerobic gasses [51]. Gas supply can be provided by surface gassing as well as submerged dip or sparging tubes which directly transfer the gas into the medium, thereby increasing the surface for gas exchange by introducing bubbles [51]. However, introducing bubbles combined with agitation and the protein composition of the medium can facilitate foam formation [56]. Amongst others, foam formation in bioreactors is associated with reduced working volumes, loss of medium and cells, as well as increased contamination risk [57]. Thus, it is important to detect and control foam. Detection of foam is usually achieved by using a probe located in the bioreactor’s top part. It measures the electric resistance, that is changing once the foam is reaching the probe [56]. It can also be used to release surface-active antifoam substances. However, these newly introduced components within the medium might change the microbe’s metabolism, reduce oxygen transfer, or impact downstream processes and upscaling [56]. Therefore, testing the antifoam with the target cell and medium of choice beforehand or using mechanical antifoam solutions is recommendable. Furthermore, to reduce foam formation in the first place, agitation and gassing should be balanced with the culture’s aeration needs. Once the culture conditions are set, further steps to support and monitor optimal culture growth can be undertaken. One of them is shortening the lag-phase after inoculating the main culture. This is achieved by (i) the right growth phase indicated by the optical density at 600 nm (OD600) within the pre-culture, (ii) the correct initial OD600 in the main culture after inoculation with the pre-culture and (iii) the right volumetric amount of bacterial pre-culture within the main culture. According to the literature, E. coli inoculation pre-cultures from shake flasks are grown to OD600 of 6-15 [58, 59]. As mentioned earlier, these values are within the range of exponential growth and ensure actively expanding bacteria [46]. When inoculating the main culture within the bioreactor, the pre-culture gets diluted to initial OD600 between 0.04 and 0.4, as described for E. coli in various settings [58, 59, 60, 61, 62, 63]. At this point, it is also important to keep the inoculation volume relatively high. 0.1-10 % of the total working volume is described in the literature but the ideal ratio should be determined by testing [45, 46, 58, 64]. After preparing the main culture this way, regular OD600 measurements should be carried out during cultivation, for example, to identify the right feeding or termination time points. Sample-taking for OD600 measurement can be performed by special sampling valves within the bioreactor and even non-invasive options exist to reduce the contamination risk even more. These tips should enable an efficient start for bioreactor runs for small-scale and scale-up processes. Scale-up Scale-up is the process of increasing the fermentation size from a small scale consisting of several milliliters to several liters to an industrial production scale with thousands or even millions of liters [65]. In principle, the same parameters important for small-scale approaches also apply to largescale, such as consistent temperature, pressure, dissolved oxygen set point, or feeding solution sterilization [64]. However, scaling up is not a one-step process of simply enlarging bioreactors and volumes. Many parameters are influenced simultaneously during scale-up which can result in inhomogeneous culture conditions. Larger bioreactors with higher volumes lead to longer mixing times with the risk of impaired oxygen and substrate distribution. Additionally, the stronger hydraulic pressure can influence the oxygen transfer rate, a critical parameter for air-to-liquid oxygen delivery. Such O2 and nutrient gradients can induce metabolic shifts within the cells, resulting in the accumulation of unwanted by-products, such as lactate or succinate which in turn affect the medium’s pH value [66]. Furthermore, high-density microbial cultures can produce significant amounts of heat which combined with inefficient mixing can lead to zonal overheating and further stress on the cells [64,66]. Thus, instead of moving immediately from small volumes to the endpoint, intermediate steps are introduced before the production scale is reached (Figure 14). Small and bench scale with volumes up to about 10 L are translated into so-called pilot scales with 1-10% of the full production scale volume [65]. Once sufficient yields are achieved in the pilot scale, translation into the manufacturing scale can be initiated. WHITE PAPER I No. 89 I Page 11 Figure 14: Illustration of a scale-up process in bioreactors. In the process of translating small (left) into production scale (right), intermediate steps, such as bench scale and pilot scale approaches, are introduced to ensure consistent bioprocess performance between the different bioreactor dimensions. During this process it is crucial to ensure reproducibility between the different scales. To this end, different scale-up parameters were defined over time, all with the goal of overall consistent culture conditions for the different scales. Such parameters include constant agitation power input per volume, oxygen transfer rate and oxygen transfer coefficient (kLa), heat transfer rate per volume, mixing time, impeller tip speed, gas volumetric flow rate, and gas superficial velocity, to name a few [64, 67]. However, due to the bioreactor’s physical properties it is not possible to keep all the parameters constant at the same time, as they can influence one another. Therefore, a selection of these parameters should be tested over all scale sizes. Out of the above, constant agitation power input per volume, constant oxygen transfer rate and constant heat transfer rate per volume are among the commonly used ones in large-scale microbial fermentation [64]. The agitation power consumption of an impeller is influenced by the agitation speed (rpm), the medium’s viscosity (kg/m3 ), the impeller’s outer diameter (m), and a dimensionless power number [64]. The power or Newton number is experimentally determined for a given impeller by measuring its torque but can also be obtained from standard impeller references or vendors [64,67,68]. Once all these variables are obtained, the final power consumption per volume given in W/m3 can be calculated by a specific mathematical formula [64, 68]. The oxygen transfer rate (OTR), given in mmol O2 /(L x h), is a measure of the gas-to-liquid transfer efficiency of oxygen during gassing. It is influenced by the agitation speed, airflow rate, pressure, and pure oxygen supply [64]. Just as the power consumption per volume, the OTR requires experimental determination as well. Different methods exist, for example the sulfite depletion method. Here, the oxygen-driven reaction of Na2 SO3 to Na2 SO4 is used to determine how fast oxygen is replenished after it was absorbed by sulfite within the medium [69]. With these results the oxygen transfer rate for a specific bioreactor can be calculated. Furthermore, oxygen transfer rates can be obtained from equipment manufacturers. However, as these values often represent the possible maximum capability of a bioreactor it should be considered to perform own experiments for a given setup [69]. As mentioned already, high-density cell cultures can produce large amounts of heat which make the heat transfer rate an important factor for keeping temperatures controllable. It is influenced by the area covered by the cooling device, as well as the culture’s and cooling agent’s temperature [64]. Besides parameters directly linked to the bioreactor, also other things need to be considered when planning a scale-up process. Deep freeze cell banks for storing backups of bacterial strains as well as the necessity of seed trains or bioreactor plant stress tests need to be considered [65, 64]. As shown by these examples, scale-up processes are complex and lengthy. Transitioning from lab to production scale WHITE PAPER I No. 89 I Page 12 can take several years [65]. Nevertheless, it is important not to rush it. The scale-up should be planned out with the final goal in mind and all your actions should be positioned towards this goal. A detailed road map should be created beforehand, containing considerations like timelines, costs, unit operation design, energy-balance, risk assessment and mitigation plans [65]. Especially during this early stage of planning, input from experts with scale-up experience and inclusion of such expertise within your team are valuable [65, 64]. Once the foundation is laid, the initial ideas should be regularly adapted according to small, bench and pilot scale experimental results. Furthermore, the smaller scale approaches should always be designed in a way to represent the larger scales as closely as possible to make the larger scales as reproducible as possible [65]. In summary, there is no one-fits-all solution for upscaling. Many parameters need to be evaluated for your individual approach. However, this extra work will pay by receiving consistent yields from benchtop to production scale. 4) Culture harvesting Small scale cultures from shake flasks or small fermenters are typically harvested by centrifugation. A benchtop multipurpose centrifuge with swing-bucket rotor is typically used for harvesting from conical tubes or conical bioreactors. A typical E. coli pelleting run is performed at around 5,000 g for ~10–20 min at room temperature or 4 °C. If higher g-forces are required to reduce the harvesting time or for harvesting larger volumes, fixed-angle rotors are the appropriate choice. The available purification system protocol usually gives a good guideline on the centrifugation settings. If the recommended centrifugation time or speed is exceeded, the cell pellet may be more difficult to resuspend. On the other hand, insufficient centrifugation time or speed may result in incomplete pelleting of cells and loss of starting material. After pelleting the cells and removal of the supernatant, the cells can be stored at -20°C for subsequent plasmid isolation. Larger industrial-scale fermenter cultures are typically harvested by either centrifugation or by tangential flow filtration (TFF) [70]. Besides conventional centrifuges, other types such as continuous-feed centrifuges can be used for the harvesting step [71]. 5) Purification The process of DNA extraction and purification consists of five basic steps: 1) cell lysis, the disruption of the cellular structure to create a lysate 2) separation of the soluble DNA from cell debris and other insoluble material, 3) binding of desired the DNA to a purification matrix, 4) washing step to remove cellular contaminants from the matrix, 5) elution of the DNA. The primary consideration for plasmid purification is separation of plasmid DNA from the host bacterium’s chromosomal DNA and cellular RNA. Besides these components, other contaminants such as proteins, salts, and endotoxins are removed as well. Furthermore, the process aims towards eliminating undesired plasmid isoforms in the final product, as plasmids may exist in supercoiled, open circular and linear configuration, resulting from conformational changes within the bacterial host cell, or during the purification process [72]. However, the supercoiled structure is considered the native and active plasmid form and mostly the desired conformation. To generate a cleared lysate with free plasmid DNA, several methods have been developed, including SDSalkaline denaturation [73,74], salt-SDS precipitation [75], or rapid boiling [76]. SDS-alkaline denaturation is a popular procedure for purifying plasmid DNA because of its overall versatility and consistency. This technique exploits the difference in denaturation and renaturation characteristics of covalently closed circular plasmid DNA and chromosomal DNA fragments. Under alkaline conditions (pH 11.5-12.5) [77], both plasmid and chromosomal DNA are efficiently denatured. Rapid neutralization with a high-salt buffer, such as potassium acetate, in the presence of SDS has two effects that contribute to the overall effectiveness of the method. First, the rapid neutralization causes the chromosomal DNA to base-pair in an intrastrand manner, forming an insoluble aggregate that precipitates out of solution. The covalently closed nature of the circular plasmid DNA promotes interstrand rehybridization, allowing the plasmid to remain in solution. Second, the potassium salt of SDS is insoluble, so the proteins and detergent precipitate and aggregate, which assists in the entrapment of the high-molecular-weight chromosomal DNA. Accurate execution of SDS-Alkaline lysis is critical for sufficient plasmid yields. First, the lysis pH of ~11.5-12.5 is close to the denaturation pH of pDNA. Thus, small pH deviations can favor the formation of undesired circular, single stranded plasmid DNA isoforms. Lysis time should be long enough for complete cell lysis but short enough to not denature pDNA. Second, aggressive mixing may lead to degrading of pDNA due to excessive shear forces, while insufficient mixing may lead to incomplete cell lysis. Also, ensuring complete neutralization of the solution during the neutralization step is critical. For research scale kit-based purification, adhere to the given times in the supplier’s protocol. WHITE PAPER I No. 89 I Page 13 The separation of soluble and insoluble material is accomplished by a clearing method (e.g. filtration, magnetic clearing, or centrifugation). The soluble plasmid DNA is ready to be further purified. There are several methods available to purify plasmid DNA from the cleared lysate, including: > binding plasmid to silica in the presence of high concentrations of chaotropic salts [78,79,80] > differential precipitation of plasmid DNA from aqueous chaotropic salt/ethanol solutions [81,82,83] > ion exchange chromatography over DEAE-modified cellulose membranes [84] > precipitation with polyethylene glycol (85,86) organic extraction using phenol [87] These methods can be adapted to various scales, from centrifugation-based minipreps to large-scale automated and semi-automated methods. The volume of the bacterial culture should match the distinct isolation system. For research scales, spin column based mini, midi or maxi preps provide fast and easy purification usually together usually with a fixed-angle microcentrifuge. Typical volume ranges and yields from Promega [88] plasmid preparation kits are listed (Table 4). Some suppliers provide Mega and Giga preps, that allow purification from even larger volumes up to 5 L with yields up to 10 mg [16]. Table 4: Typical volume ranges and yields with Promega plasmid purification kits for laboratory scale purifications (Source: Promega). Please note that kits from other suppliers can vary. Culture size Sample volume Isolation system Average pDNA yield high copy plasmid Small 0.6-3 mL Miniprep 1.5–15.0 μg Medium 50–100 mL Midiprep 100–200 µg Large 250 mL Maxiprep Up to 1 mg For low volume, high-throughput processing, isolation systems based on a 96-well format are available based on vacuum or magnetic beads. Methods used at small scale are not readily scalable for large scale. For large-scale pDNA production, the steps after alkaline lysis are followed typically by different filtration techniques or centrifugation with a final chromatography to separate pDNA from residual impurities [72]. The biomass should be kept in a range that is acceptable for the plasmid isolation system used. Most research scale spin column based purification systems are optimized for use with LB media [88,89]. Using enriched media like TB that leads to higher biomasses per liter, that may cause an overloading of the purification system and insufficient lysis [16,88]. The culture volume per column should be reduced in these cases to match the recommended biomass. That also applies to extremely high copy plasmids and host strains that show high growth rates. Furthermore, the potential higher viscosity of the lysate will require more vigorous mixing, which may result in shearing of bacterial genomic DNA and contamination of the plasmid DNA [16]. For very high-sized plasmids (> 20,000 bps) a reduced efficiency in elution from the column is noted, the limitation in size should be checked in the manual of the used supplier [16,88]. pDNA concentration in a laboratory scale is typically determined by measuring the absorbance at 260 nm (A260) in a spectrophotometer, for example using a quartz cuvette. Standard spectrometers allow measurement of the A260/230 and the A260/280 ratios to provide valuable information on the plasmid DNA’s purity. 1) Determination of the purity ratios A260/A280 reveal nucleic acid sample quality and possible contamination with protein, phenols or other aromatic compounds. A ratio of 1.8-2.0 indicates sufficient pDNA quality [89] 2) The A260/A230 ratio reveals nucleic acid sample quality and possible contamination with organic compounds. This ratio should be > 2.0 [89]. 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A procedure for the large-scale isolation of highly purified plasmid DNA using alkaline extraction and binding to glass powder. Anal Biochem. 1982 Apr;121(2):382-7 WHITE PAPER I No. 89 I Page 17 www.eppendorf.com Ultra Yield® and Plasmid+® are registered trademarks of Thomson Instrument Company, USA. Promega® and pGEM® are registered trademarks of Promega GmbH, Germany. Eppendorf®, the Eppendorf Brand Design, and Eppendorf Tubes® are registered trademarks of Eppendorf SE, Hamburg, Germany. All rights reserved, including graphics and images. Copyright © 2023 by Eppendorf SE. Your local distributor: www.eppendorf.com/contact Eppendorf SE · 22331 Hamburg · Germany eppendorf@eppendorf.com · www.eppendorf.com About Eppendorf Since 1945, the Eppendorf brand has been synonymous with customer-oriented processes and innovative products, such as laboratory devices and consumables for liquid handling, cell handling and sample handling. 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