Engineering Commons^™

Continuous Process Performance Enhancements For 50 L To 500 L Single-Use Bioreactors: A Technical Comparison Of Erformance Characterization, Cell Culture, And Scale-Up Modeling, Tony Hsiao, Nephi Jones, Jordan Cobia

Integrated Continuous Biomanufacturing IV

Improvements in single-use systems have allowed implementation of high-density cultures in emerging bioprocess work flows. Specifically, advantages of single-use bioreactors have been realized in perfusion applications in high-density seed train intensification or as a compact production-scale bioreactor system. Due to this and additional progressive advances in media optimization and improved clone genetic selection have increased stress on the perceived performance limitations of single-use bioreactors. This study shows integration of the Thermo Scientific™ HyPerforma™ Single-Use Bioreactor (S.U.B.) and how strategic enhancements to the sparger and agitation systems have revealed the potential for 3-4X improvement of mixing and mass transfer performance compared …

Hydrocyclones For Single-Use Perfusion Application, Ioná W. Bettinardi, Andreas Castan, Ricardo A. Medronho, Leda R. Castilho

Integrated Continuous Biomanufacturing IV

Hydrocyclones (HC) are very compact devices that promote solid-liquid separation under the action of a centrifugal field. Despite the small size, HCs have a large processing capacity and do not suffer from clogging. Therefore, several publications explored HCs as a potential cell retention device in perfusion applications in the last 20 years, but limited to non-disposable lab-scale bioreactors and to relatively low cell densities (up to ~10 million cells/mL).

Even though the absence of moving parts may streamline the HC manufacturing, the performance of solid-liquid separation is highly dependent on the HC internal geometry. Said that, hydrocyclones can be produced …

Production Of Zika Virus-Like Particles (Vlps) By Perfusion Processes, Renata G. F. Alvim, Leda R. Castilho

Integrated Continuous Biomanufacturing IV

Zika virus (ZIKV) emerged as a major international public health concern in 2015 and rapidly spread to more than 80 countries in Africa, Asia and the Americas. ZIKV infection has been shown to cause Guillain-Barré syndrome in adults, as well as severe congenital malformations in fetuses from as much as 42% of infected mothers (Brasil et al., 2016, doi:10.1056/NEJMoa1602412). While no ZIKV vaccine becomes approved for human use, periodic outbreaks will continue to occur in endemic regions and the risk of spreading to non-endemic regions will continue to exist, especially because ZIKV persists in body fluids for very long time …

Scalable Lentiviral Vector Production Using Stable Producer Cell Lines In Perfusion Mode, Sven Ansorge, Aziza Manceur, Stéphane Lanthier, Sonia Tremblay, Anne-Marie Gélinas, July Dorion-Thibaudeau, Sophie Broussau, Julia Transfiguracion, Hafida Aomari, Rénald Gilbert

Cell Culture Engineering XVI

Lentiviral vectors (LVs) are becoming an important tool in gene and cell therapy and are being utilized in several clinical studies against genetic and acquired diseases, as well as in cancer therapies. To address the challenges linked to the generation of preclinical and clinical supply, the National Research Council Canada has developed packaging cell lines and stable producer cell lines for the production of LVs which can grow in suspension in serum-free media and produce LV in the 106 TU/ml range without optimization. We focus on the development of perfusion processes to both intensify the process and harvest produced LV …

Ultra Scale-Down Mimics For Perfusion Culture: Experimental Study For Rapid Biopharmaceutical Process Development, Molly Tregidgo, David Pollard, Martina Micheletti

Integrated Continuous Biomanufacturing III

With the industry driving towards the implementation of whole continuous bioprocess sequences, there becomes a requirement for the development of scale-down tools with the same consistency, reliability and throughput as those already available for traditional batch processes. This work aims to develop a perfusion scale-down system capable of reproducing the specific characteristics of the perfusion culture process, namely cell retention capabilities, the ability to support high cell densities and to operate for extended periods compared to fed-batch cultures. Cell culture in microwell plates in fed-batch mode is well defined and is in widespread use; however to the best of our …

Fouling Mitigation In Membrane Based Perfusion Systems By Oscillating Tangential Flow, Maria Weinberger, Ulrich Kulozik

Integrated Continuous Biomanufacturing III

Good scalability and robust handling have promoted the application of membrane based cell retention devices. A physical barrier, i.e. a filter, retains cells and cell debris. One major drawback of these devices is their ten-dency to foul and clog. One form of fouling is deposit layer formation on the filter surface, consisting of cells, cell debris and other fermentation broth constituents. This leads to the build-up of a secondary membrane, which can alter the permeation profile. Furthermore, deposit layers lead to an increased filtration resistance and thus negatively affect permeate flux, filtration efficiency and process robustness. In tangential flow filtration, …

Up And Down Scale Considerations For The Continuous Production Of Glycooptimized Biopharmaceuticals, Steffen Kreye, Rainer Stahn

Integrated Continuous Biomanufacturing III

Product quality especially with respect to glycosylation is a critical product attribute which can have an immense influence on the activity of glycoproteins and should be closely monitored during process development and manufacturing. GlycoExpressTM cells have been developed in this context to provide human host cell lines with robust glycosylation machineries. This work will address challenges in two case studies for up and down scaling of production processes with focus on product quality and continuous processing. In a first section, data of a process transfer from a 200 L stainless steel to a 1000 L single-use bioreactor for a continuous …

Bioprocess Intensification And Optimisation Using Macroscopic Predictive Models Of Cell Culture Processes, Bassem Ben Yahia, Boris Fessler, Gwendal Gränicher, An-Vy Tran, Mareike Harmsen, Elmar Heinzle

Integrated Continuous Biomanufacturing III

Chinese Ovary Hamster (CHO) are an indispensable tool for biotechnological production of biologics which is a multi-million business. Recently, the pharmaceutical industry is increasingly focusing in the use of perfusion mode. Nevertheless, the optimal perfusion rate during biopharmaceutical perfusion production is dependent on cell metabolism which can be characterized by mathematical models. This study provides insights into the predictive capacities of systematic and simple cell modeling approaches of metabolism, growth and production of monoclonal antibodies (mAb) [1] to optimized medium composition and perfusion rate during CHO perfusion culture. We applied the metabolic steady state concept and used a segmented linear …

Fouling Mitigation In Membrane Based Perfusion Systems By Oscillating Tangential Flow, Maria Weinberger, Ulrich Kulozik

Integrated Continuous Biomanufacturing III

Good scalability and robust handling have promoted the application of membrane based cell retention devices. A physical barrier, i.e. a filter, retains cells and cell debris. One major drawback of these devices is their ten-dency to foul and clog. One form of fouling is deposit layer formation on the filter surface, consisting of cells, cell debris and other fermentation broth constituents. This leads to the build-up of a secondary membrane, which can alter the permeation profile. Furthermore, deposit layers lead to an increased filtration resistance and thus negatively affect permeate flux, filtration efficiency and process robustness. In tangential flow filtration, …

Ultra Scale-Down Mimics For Perfusion Culture: Experimental Study For Rapid Biopharmaceutical Process Development, Molly Tregidgo, Martina Micheletti, David Pollard

Integrated Continuous Biomanufacturing III

With the industry driving towards the implementation of whole continuous bioprocess sequences, there becomes a requirement for the development of scale-down tools with the same consistency, reliability and throughput as those already available for traditional batch processes. This work aims to develop a perfusion scale-down system capable of reproducing the specific characteristics of the perfusion culture process, namely cell retention capabilities, the ability to support high cell densities and to operate for extended periods compared to fed-batch cultures. Cell culture in microwell plates in fed-batch mode is well defined and is in widespread use; however to the best of our …

Up And Down Scale Considerations For The Continuous Production Of Glycooptimized Biopharmaceuticals, Steffen Kreye, Rainer Stahn

Integrated Continuous Biomanufacturing III

Product quality especially with respect to glycosylation is a critical product attribute which can have an immense influence on the activity of glycoproteins and should be closely monitored during process development and manufacturing. GlycoExpressTM cells have been developed in this context to provide human host cell lines with robust glycosylation machineries. This work will address challenges in two case studies for up and down scaling of production processes with focus on product quality and continuous processing. In a first section, data of a process transfer from a 200 L stainless steel to a 1000 L single-use bioreactor for a continuous …

Bioprocess Intensification And Optimisation Using Macroscopic Predictive Models Of Cell Culture Processes, Bassem Ben Yahia, Mareike Harmsen, An-Vy Tran, Gwendal Gränicher, Boris Fessler, Elmar Heinzle

Integrated Continuous Biomanufacturing III

CHO cells, modeling, perfusion, ATF, segmented linear model

Optimizing Media For Perfusion Combining Predictive Scale-Down Models And Multivariate Approaches, Jochen Sieck, Christian Schultheiss, Pedro Felizardo

Integrated Continuous Biomanufacturing III

Perfusion medium development is complex and time-consuming. Thus, a methodology for efficient and cost effective development leading to a significant reduction of time to market is key in today's competitive market. Success in perfusion medium development depends strongly on the choice of optimization tools. Here a streamlined strategic approach to accentuate perfusion media development is presented by means of predictive scale down models and the application of Design of Experiments (DOE). The purpose of the study was to investigate the impact of 19 amino acids on productivity and growth of a CHO-K1 cell line in perfusion cell culture. A stepwise …

Toward Development Of Continuous Bioprocesses: Comparison Of Fed-Batch And Perfusion Upstream Production Processes In Early Development, Jean Mclarty, Daryl Powers, Christine Hamel, Betsy Simons, Ken Karey

Cell Culture Engineering XV

Continuous Processing is an exciting development in the field of bioprocessing. The potential for quick response to market demands, decrease in infrastructure, increased flexibility and consistent product quality has resulted in a growing interest in Continuous Processing for production of all types of protein drugs (high or low volume, stable or unstable). Sanofi is developing a novel Integrated Continuous Manufacturing platform for biologics that utilizes an upstream perfusion process. While cell culture perfusion processes offer substantial benefits for commercial biologics production, implementation may present challenges in early development, where speed to first in man studies is critical. Here we present …

Non-Invasive Real-Time Monitoring Of Glucose And Lactate By Nir-Spectroscopy During Perfusion Cho Culture, Jean Hamel, Anita Wamakima, Ruifan Pei

Cell Culture Engineering XV

Near-infrared spectroscopy (NIR) has been used to monitor glycerol and methanol, non-invasively during cultures of Pichia pastoris^{1, 2}. In this work, glucose and lactate were measured in Chinese Hamster Ovary (CHO) perfusion culture, in real time, using an online advanced NIR monitor and a reference offline biochemical analyzer. The 1.8-L culture started in the batch phase at 4 g/L glucose with 0.3 x 10⁶ cells/mL and reached 1.5 x 10⁶ cells/mL after 90 hours. Perfusion was then initiated and conducted for 10 days at 0.7 vvd, using a spiral membrane-less microfluidic device³. The maximum …

Process Optimization For Semi-Continuous Virus Production At High Cell Densities, Daniel Vazquez

Cell Culture Engineering XV

Background. Unlike production of recombinant proteins, continuous production of viral vaccines at high cell densities (HCD) is still constrained by host cell lysis during virus propagation and limited virus recovery from culture broth. Nevertheless, advanced fed-batch [1] and perfusion strategies can be applied to achieve a high-yield virus production processes. In this study, the development of a high-yield semi-continuous process for the production and purification of the modified vaccinia Ankara virus isolate MVA-CR19 and influenza A/PR/8 in HCD cultivations of the suspension cell line AGE1.CR.pIX (ProBioGen AG, Berlin) is presented.

Methods. Depending on the required scale, high cell …

Size Matters: Assessment Of A Larger Pore Hollow Fiber To Reduce Product Retention In Perfusion, Samantha Wang

Cell Culture Engineering XV

Traditionally, perfusion processes using either TFF or ATF technologies have utilized hollow fibers made from polymers such as polyethersulfone (PES) or polysulfone (PS) for cell retention. The pore sizes on these hollow fibers range from several hundred kD to a few µm. However, despite the relatively large size of the pores compared to that of the product, retention of product across the hollow fiber over time is a known and common problem in perfusion. Particle size analysis demonstrated accumulation of particles around 100nm in diameter, which are unable to pass through a typical 0.2µm PES membrane. As these particles are …

Bioreactor Process Improvements In A Legacy Perfusion-Based Process, Rakesh Motani, Gonzalo Milet, Gregory Walsh, Lada Laenen

Cell Culture Engineering XV

The legacy manufacturing processes for production of the enzyme products at Genzyme include long-term bioreactor perfusion-based cell culture platform. For the process with tight filed ranges and limited characterization, a phased approach is being used to improve bioreactor productivity. In the first phase, short-term process changes that are within filed and historical ranges were implemented. In the second phase, long-term process improvements that are outside the filed ranges will be implemented for a significant improvement in bioreactor productivity. Results from lab and at-scale study have confirmed that there was no adverse impact of phase 2 process improvements on cell culture, …

Implementation Of A Recirculating Tff N-1 Perfusion System At Manufacturing Scale: Conquering Process Hurdles And Scaling Challenges, Alex Doane, Weiwei Hu, William Yang, Kelly Wiltberger, Haofan Peng

Cell Culture Engineering XV

In order to increase the output of a traditional large scale stainless steel fed-batch manufacturing facility without major engineering work, we investigated the implementation of N-1 perfusion technology to feed a more intensified fed-batch production process. Perfusion N-1 enables cells to grow to high densities in the seed train bioreactor. By shifting cell growth from production to seed train, we can increase seed train occupancy per batch and reduce production bioreactor occupancy per batch, which improves facility throughput by bringing the run duration ratio of N-1/N closer to unity. Instead of using the popular ATF technology, an in-house TFF system …

Efforts To Reduce Impact Of Media Variability On Product Quality For A Commercial Perfusion Process, Nirel Rillera, Kevin Yeh, Benjamin Youn, Jerome Acuna, John Henstrand, Sean Forestell

Cell Culture Engineering XV

Strict control of raw materials used in bioprocesses is necessary to ensure consistent product quality. However, use of poorly characterized complex components makes achieving such strict control difficult. A strong correlation between product quality variability among bulk drug substance lots and changes in a complex medium component was observed in a commercial perfusion process. This correlation was subsequently confirmed experimentally at both large and small scales. A multifaceted strategy was applied to investigate and mitigate the impact of the media variability on product quality, which included (1) studies in small scale perfusion bioreactors; (2) development of a simple cell based …

Cell-Controlled High-Intensity Perfusion And Hybrid Fed-Batch Systems That Drastically Reduce Perfusion Rates And Harmonize With Continuous Downstream Processing, Gregory Hiller, Matthew Gagnon, Ana Maria Ovalle, Bhanu Chandra, Wenge Wang, Elizabeth Eydelman

Cell Culture Engineering XV

Peak cell densities in fed-batch cultures of mammalian cells can be limited by the depletion of nutrients or accumulation of inhibitors. Since addition of nutrients is relatively straight forward, inevitably it is growth inhibitor buildup, combined with the practical limits of feed volume addition and the problem of amino acid counter ion and miscellaneous osmolyte accumulation, that serve to limit productivity in the industry standard fed-batch bioreactor. To break these barriers we have begun to investigate mixed modes of bioreactor operation, some using novel methods for cell-controlled perfusion, which can substantially reduce the volumes of perfusion medium required, suppress lactic …

Overcoming Process Intensification Challenges To Deliver A Manufacturable And Competitive Integrated Continuous Biomanufacturing Platform, Jason Walther, Neha Shah, Myles Hollenbach, Jonathan Wang, Marcella Yu, Jiuyi Lu, Yang Yang, Konstantin Konstantinov, Chris Hwang

Cell Culture Engineering XV

Groups in both industry and academia have achieved high densities and productivities in perfusion cell culture processes. At Sanofi, we have demonstrated perfusion densities greater than 100 million cells/mL (with associated high productivities) at a cell-specific perfusion rate of only 20 pL/cell/day. This process intensification reduces the footprint of upstream unit operations as well as capital and operating expenses of manufacturing facilities. The continuous nature of perfusion cell culture also creates opportunities for integration of continuous downstream operations, leading to further process intensifications and volume reductions.

In this presentation, we will discuss our work on several upstream challenges that must …

Evolution Of An Integrated Continuous Antibody Manufacturing Process, Christopher Kistler

Cell Culture Engineering XV

Efforts continue to reduce the timeline and cost of progressing biologics from discovery through preclinical development to product launch. Integrated processes, those that link upstream and downstream processing with control of product quality are an attractive way to achieve meaningful reductions in the overall cost of ownership of a biologics manufacturing process.

To start along this path, an integrated continuous processing laboratory, the PROLab (Protein Refinery Operations Lab) was built to pilot the concepts of an integrated continuous antibody manufacturing process under automated control. A perfusion process was developed capable of operating for >30 days using either TFF (Spectrum KrosFlo® …

Process Optimization For Semi-Continuous Virus Production At High Cell Densities, Daniel Vazquez, Michael Pieler, Yvonne Genzel, Ingo Jordan, Volker Sandig, Udo Reichl

Cell Culture Engineering XV

Background. Unlike production of recombinant proteins, continuous production of viral vaccines at high cell densities (HCD) is still constrained by host cell lysis during virus propagation and limited virus recovery from culture broth. Nevertheless, advanced fed-batch [1] and perfusion strategies can be applied to achieve a high-yield virus production processes. In this study, the development of a high-yield semi-continuous process for the production and purification of the modified vaccinia Ankara virus isolate MVA-CR19 and influenza A/PR/8 in HCD cultivations of the suspension cell line AGE1.CR.pIX (ProBioGen AG, Berlin) is presented.

Methods. Depending on the required scale, high cell …

Exploring Continuous And Integrated Strategies For The Up- And Downstream Processing Of Human Mesenchymal Stem Cells, Barbara Cunha, Tiago Aguiar, Ricardo Silva, Cristina Peixoto, Manuel Carrondo, Margarida Serra, Paula Alves

Integrated Continuous Biomanufacturing II

The integration of up- and downstream unit operations can result in the elimination of hold steps, thus decreasing the footprint, and ultimately can create robust closed system operations. This type of design is desirable for the bioprocess of human mesenchymal stem cells (hMSC), where high numbers of pure cells, at low volumes, need to be delivered for therapy applications. The aim of this work is to perform a proof of concept of the integration of a continuous perfusion culture in bioreactors with a tangential flow filtration (TFF) system for the concentration and washing of hMSC. In particular, we have evaluated …

Protein Refinery Operations Lab (Pro Lab): A Sandbox For Continuous Protein Production & Advanced Process Control, Mark Brower, David Pollard, Finn Hung

Integrated Continuous Biomanufacturing II

Significant strides towards implementation of continuous bioprocessing are being made at an ever increasing rate. Advances in technology for traditional unit operations such as cell-retention devices in perfusion cell culture, continuous multi-column chromatography (CMCC) and single-pass tangential flow filtration have led to demonstrations of both semi-continuous and fully-continuous protein production processes operating at periodic steady states at the pilot-scale. Previous proof of concept work at Merck & Co., Inc. has shown an automated (DeltaV) and single-use monoclonal antibody (mAb) purification scheme through Protein A CMCC and pH viral inactivation with minimal human interaction for 30 days fed from a perfusion …

Small-Scale Platform For Rapid On-Demand Manufacturing Of Recombinant Proteins, J. Christopher Love

Integrated Continuous Biomanufacturing II

The current timeline for manufacturing high-quality recombinant proteins used for therapeutics and vaccines, and delivering them to patients, typically requires many months. There are many circumstances, however, where the rapid production, release, and delivery of these biologics could address unmet medical needs, including shortages, emergency situations, and pandemics. This talk will present a new platform developed to produce biologic medicines on demand called Integrated and Scalable Cyto-Technology (InSCyT). This platform comprises an integrated, subliter-scale portable system for the (semi)continuous operation of fermentation, filtration of cell debris from secreted product, innovative affinity-based purification, polishing, and finishing. This program also emphasizes integrated …

From Fed-Batch To Perfusion: Productivity And Quality Considerations For A Late-Stage Program, Sen Xu, Hao Chen

Integrated Continuous Biomanufacturing II

In this poster, we will present the rapid development of a perfusion process with high productivity for late-stage manufacturing. The process uses a Chinese Hamster Ovary (CHO) cell line for monoclonal antibody production. Prior to the development, the clinical supply was manufactured by a fed-batch process with the same cell line.

To achieve desired productivity and comparable quality attributes, we developed a repeated batch shake flask model to rapidly screen media and process conditions. Shake flasks were inoculated at 10 × 10⁶ cells/mL and maintained in a CO₂ incubator with medium exchange for every 48 hours, repeated for …

Designing A Microbial Cultivation Platform For Continuous Biopharmaceutical Production, Nicholas Mozdzierz, Kerry Love, Alan Stockdale, John Clark, Noelle Colant, Christopher Love

Integrated Continuous Biomanufacturing II

The existing biopharmaceutical manufacturing paradigm is poorly suited to produce biologic drugs on demand at a point-of-care. Generally, commercial-scale (~2,000 - 10,000 L) manufacturing using fed-batch cultivation and fixed stainless-steel infrastructure is concentrated in developed nations and results in process cycle times on the order of weeks to months.^1,2 Coupled with the complex logistical challenges associated with continuous “plant-to-patient” cold-chains, the geographically biased nature of therapeutic protein production today can limit access to biologic drugs in developing areas of the world.³ There is an opportunity to create technologies capable of rapidly generating biopharmaceuticals in situ in emergency situations, …

Integrated Solutions For Continuous Processing In Mobius® Bioreactor Systems, Andrew Clutterbuck

Integrated Continuous Biomanufacturing II

Throughout the bioprocess industry, the use of disposable systems such as bioreactors is playing an increasingly significant role. Manufactures are recognising the strategic benefits of single use systems in both reducing production costs and increasing overall manufacturing throughput.

Over the past few years there has been increasing demand for larger scale disposable bioreactor systems, to satisfy the manufacturing demands and an emphasis on suppliers such as Merck Millipore to demonstrate robust and reproducible scale-up within these systems.

This presentation will explore the design philosophies behind the Mobius^® bioreactors and will also give an overview of the novel SensorReady external …