Engineering Commons^™

Integrated Continuous Processing For The Manufacture Of Monoclonal Antibodies, Daniel Karst, Fabian Steinenbach, Massimo Morbidelli

Integrated Continuous Biomanufacturing II

Continuous manufacturing is currently being considered by the Biopharmaceutical Industry not only for the classical reasons which make continuous operation preferred over the batch one, but also for recent initiatives of the regulatory agencies. We discuss here a series of experiments where a perfusion reactor with CHO cells for the production of a monoclonal antibody has been operated in the continuous mode and connected to a two column continuous protein A chromatographic unit for product capture. A few steady states are examined and the use of simulation models for process design and control is illustrated.

Integrated Continuous Bioprocessing – Opportunities And Challenges, Mats Akesson, Martin Heitmann, Ditte Skipstrup, Peter Tiainen

Integrated Continuous Biomanufacturing II

We have developed an integrated continuous processing framework for end-to-end production of complex fragile proteins based on perfusion cultivation and automated multi-step purification [1,2,3]. Upstream, the integrated system consists of a stirred tank bioreactor with an ATF cell retention system. The clarified harvest directly enters an off-the-shelf ÄKTA chromatography system converted into a continuous purification unit. Two alternating capture columns precede an automated multi-step purification train with full flexibility and control of individual columns.

The integrated set-up enables compact automated bench-top factories converting cell culture media to purified protein in an efficient manner without intermediate storage. It provides monitoring of …

Continuous Culture And Downstream Processing Of Algae With Recycle: An Integrated Large-Scale Approach For Production Of Renewable Crude Oil, Matt Croughan

Integrated Continuous Biomanufacturing II

In order to have a significant impact on energy security and reduction of greenhouse gasses, the production of advanced biofuels must be increased to billions of gallons per year. Scale up of phototrophic algae bioprocessing for renewable oil production has been attempted for decades and has recently reached new levels of performance and scale. For over three years, Sapphire has successfully operated the world’s largest algae farm, consisting of 100 acres of ponds in New Mexico, used to feed an integrated conversion and extraction unit based on high temperature liquefaction technology. All aspects of the process are run on a …

Continuous Beer Production Methods: A Review Of Chances And Risks, Konrad Müller-Auffermann

Integrated Continuous Biomanufacturing II

Beer brewing is one of the oldest biotechnological processes applied by humans. The production methods are comparatively complex and time consuming, which is the reason why inventors and scientists have steadily attempted to improve its manufacturing.

During the industrial revolution more and more processes where done continuously in order to increase production efficiency. Breweries where trying to modify the classical batch modes into a continuous production process but were unsuccessful. Nearly all approaches failed due to changes of the beer quality.

The presentation shall give a brief overview of different continuous production concepts tested in the past 150 years. Furthermore …

Laboratory Scale Continuous Linear Purification As A Development Tool For Recombinant Blood Protein Processing, Using Chromatographic Resins And Membranes, Michael Hughson, Rimenys Carvalho, Thaynna Araujo Cruz, Leda Dos Reis Castilho

Integrated Continuous Biomanufacturing II

Continuous processing offers significant advantages for the processing of unstable recombinant products such as therapeutic plasma proteins. As such we have established a development platform to assess potential purification steps as part of a continuous linear process using a standard AKTA Explorer. Following a consistent format of IEX membrane to HIC membrane to affinity resin, we were able to rapidly investigate multiple purification pathways for a recombinant blood protein. Using membranes as the first two steps enables a 3-step purification process to be carried out in 3 to 4 hours, with a total turnaround of 5 to 6 hours including …

Continuous Countercurrent Tangential Chromatography For Purification Of High Value Therapeutic Proteins, Andrew Zydney, Oleg Shinkazh, Boris Napadensky, Amit Dutta

Integrated Continuous Biomanufacturing II

One of the main challenges in the development of integrated continuous processes for the production of high value therapeutic proteins is the replacement of traditional column chromatography, which tends to dominate current downstream processing. Continuous Countercurrent Tangential Chromatography (CCTC) has been designed to provide truly continuous product capture and purification using a column-free system that overcomes many limitations of traditional column chromatography. All operations in CCTC are conducted on a moving slurry that is continuously pumped through a cascade of static mixers (for mixing and residence time) and hollow fiber membrane modules (for separation of the fluid phase from the …

Modeling Perfusion At Small Scale Using Ambr15tm, Dustin Davis, Delia Lyons, Scott Ross

Integrated Continuous Biomanufacturing II

Reaching cell densities higher than 80 million with the minimum possible perfusion rates is a goal for an increasing proportion of processes developed by biopharmaceutical companies. With the goal of fulfilling the industry needs for better commercial and customized perfusion media, SAFC evaluated different small-scale perfusion models to achieve an efficient work flow that can accommodate perfusion systems. SAFC successfully uses an optimized work flow for the development of media and feeds for fed-batch cell culture that integrates high-throughput screening, statistical tools and bench-top bioreactor scale studies. In this model, 96-deep well plates are used for the initial high throughput …

Monitoring Intracellular Component Pools To Identify Steady State In Mammalian Cell Perfusion Culture, Daniel Karst, Robert Steinhoff, Marie Kopp, Renato Zenobi, Massimo Morbidelli, Miroslav Soos

Integrated Continuous Biomanufacturing II

Perfusion cultures of mammalian cells are a well-considered alternative in the process development for new biologic products. Stable operation of the culture should eventually lead to a steady state, positively effecting product quality. Although macroscopic variables such as viable cell density, main metabolite or product concentrations are observed constant, little is known about the extent of steady state on intracellular level.

In this study intracellular component pools of CHO cells were monitored at different steady states in perfusion cultures using matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). A novel single extraction was used to separate metabolite, …

Biosc® Predict Simulation Software: Flexibility And Optimization Of Your Multi-Column Process, Fabien Rousset

Integrated Continuous Biomanufacturing II

For a few years now, the biopharma industry has started implementing continuous processing, and more specifically continuous chromatography, to improve process economics together with process control and understanding. A simulation / optimization software has been developed to benefit from modelization by accommodating batch process data to multicolumn processes with associated optimal operating costs and reduced footprint. The presentation will highlight how the flexibility of the BioSC® and its simulation software BioSC® Predict is used to optimize affinity, ion exchange and isocratic processes in order to decrease media and buffer consumption and save lab operating time.

Medium Optimization Case Study For Continuous Upstream Process, Rashmi Kshirsagar, Alan Gilbert

Integrated Continuous Biomanufacturing II

Based on other mature industries, continuous upstream process is a logical replacement for current fed batch operations. However most industrial medium development has focused on the biological requirements for fed batch and therefore focused on the needs of stationary phase production. There is not an a priori expectation that growth and stationary phase requirements are identical. Yet an ideal continuous upstream process requires some combination of both. An optimal continuous upstream process requires a high cell density similar to fed batch operations. There is also some minimum growth rate required in order to match the combined death and cell removal …

Alternating Flow Filtration As An Alternative To Internal Spin Filter Based Perfusion Process: Impact On Productivity And Product Quality, Guillermina Forno, Cristian Paillet, Laura Mauro, Ignacio Amadeo, Eduardo Orti

Integrated Continuous Biomanufacturing II

The application of an alternating flow filtration (ATF) system adapted to a stirred tank bioreactor as an alternative to internal spin filter was studied using CHO cells producing a fusion protein. As expected, it was possible to reach higher cell densities (maximum 50 x 10⁶ vs 20 x 10⁶ cells/ml) and achieve longer runs with the ATF device (50 days vs 30 days), since it is known that this cell retention device is less affected by fouling.

Even tough specific cell yield was similar for both systems; volumetric production of the reactor using the ATF was almost 70% …

A Novel Plant Cell Culture Platform For Semicontinous Production Of Recombinant Proteins: Butyrylcholinesterase As A Case Study, Jasmine Corbin, Bryce Hashimoto, Kalimuthu Karuppanan, Raymond Rodriguez, Somen Nandi, Brian Roberts, Amy Noe, Karen Mcdonald

Integrated Continuous Biomanufacturing II

In this paper we describe a novel biomanufacturing production platform that utilizes transgenic rice cell suspension cultures for efficient semicontinuous cell culture (SCC) production of recombinant proteins. The production platform utilizes a metabolically regulated promoter, a secretion signal peptide that enables secretion out of the cell for ease of recovery/purification, coupled with an efficient semicontinuous operational strategy that allows independent optimization of growth and production phases. In addition, long term operation (up several months¹) is possible by maintaining viable biomass within the bioreactor, thereby reducing the need for long seed trains, as well as minimizing turn-around time, CIP …

Integration Of Continuous Ethanol Precipitation And Flocculation Into Manufacturing Of Antibodies, Alois Jungbauer, Nikolaus Hammerschimidt, Peter Satzer

Integrated Continuous Biomanufacturing II

Precipitation and flocculation are optimal unit operations for continuous capture of proteins from culture supernatant. Precipitation and flocculation can be operated in a real continuous manner. The methods can be combined and after this capture step the precipitate can be stored as a concentrated solution or even as a precipitate. We have developed several precipitation/ flocculation protocols for capture of antibodies from culture supernatant. These include the combinations of CaCl₂ flocculation with either cold ethanol precipitation or PEG precipitation and octanoic acid precipitation with PEG precipitation. The precipitation conditions have been screened in microtiter plates or in case of …

Evaluation Of Novel Cex Resin For Continous Processing Of Mab Purification, Takuya Muramoto, Satoshi Murakami, Shinsuke Higuchi, Keiji Iwamoto

Integrated Continuous Biomanufacturing II

We have evaluated novel cation exchange resin and membrane which is potentially suited for continuous process in biologics manufacturing.

Typically cation exchange chromatography (CEX) is used in a bind/elute (B/E) mode for the MAb process since MAb act as cation at the buffer condition used for CEX due to the pI from neutral to weak basic. Generally, purpose of CEX chromatography is to remove aggregates and other impurities like HCP and DNA. Especially, aggregate removal is of interest to the industries. A continuous process in MAb downstream process can solve several bottlenecks in typical batch process. However its capability cannot …

Impact Of Closed And Continuous Processing On Biopharmaceutical Facility Layouts, Marc Pelletier

Integrated Continuous Biomanufacturing II

In response to the uncertainty of ever-changing product pipelines, the biotech industry has been driven to develop flexible manufacturing solutions. These facilities can mobilize quickly to provide a nimble response to sudden changes in production demands. The industry has concurrently improved its ability to assess and effectively mitigate risks to manufacturing processes by developing engineering solutions that isolate the bioprocess from the environment housing it. This presentation will illustrate how the implementation of continuous closed bioprocessing impacts the design of biopharmaceutical manufacturing facilities. The impact of closed continuous bioprocessing on facility size, capital cost and operating cost will also be …

What Are The Facility Design Requirements To Fit Biologics Pipeline Demands?, Thomas Sauer

Integrated Continuous Biomanufacturing II

Decisions to install new facilities are typically driven by pipeline or capacity demands. While extensions to existing capacity typically restrict the level of freedom to design, this is different for pipeline products. Due to numerous unknowns in the development of drug candidates in a pipeline, maintaining a large degree of flexibility in future facility design is the most widely accepted de-risking concept. Looking more closely into the development of a biologics pipeline, certain elements help to control and limit the design space while others just effectuate the opposite.

Without question the key guiding principle for these future products is to …

Design Of A Continuous Precipitation Operation For Protein Capture, Todd Przybycien, Orlando Jaquez, Robert Gronkel

Integrated Continuous Biomanufacturing II

Increasing product titers challenge chromatographic separations and play to the strengths of bulk separation techniques such as precipitation and liquid-liquid extraction. Further, bulk separation techniques lend themselves readily to true continuous operation without complex equipment. Here we describe the design of a continuous precipitation process for the capture of monoclonal antibodies from concentrated (~100 g/L) cell culture media. A continuous tubular reactor design with static mixing elements was implemented to ensure that each fluid element experienced the same mixing conditions between target and precipitant streams and the same overall fluid shear rate history. The continuous tubular format also permitted the …

A Biomanufacturing Facility Based On Continuous Processing And Single Use Technology, Jorgen Magnus, Maike Temming, Peter Schwan, Jovana Micovic, Martin Lobedam, Stefan Sievers

Integrated Continuous Biomanufacturing II

Bayers vision of the Biofacility of the Future is developed within the Mobidik project. A pilot plant with a complete production line of monoclonal antibodies from fermentation to final drug substance has been established. All parts in contact with the product are made in single use technology and the process is run as an integrated, fully continuous process. The process control system and the PAT concept are developed to achieve a high level of automation limiting the need for manual handling to a minimum. Issues related to GMP compliance are being addressed at an early stage. A detailed GMP risk …

Factors Affecting The Productivity Of 4-Column Periodic Counter Current Chromatography (4c-Pcc), Hani El-Sabbahy, Laura Fagan, Verity Nancollis

Integrated Continuous Biomanufacturing II

The advantages of continuous bioprocessing include steady state operation, reduced equipment footprint, high volumetric productivity, streamlined process flow, and reduced capital cost. These benefits have led to considerable interest in evaluating these technologies for the purposes of bioprocess intensification (Konstantinov & Cooney, 2014). It has been shown, through cost of goods modelling, that the cost of protein A is one of the most significant costs in a MAb process, and that decreasing the cost of protein A by maximising loading or increasing lifetime will have a significant effect on consumable costs (Broly, et al., 2010). Consequently, the application of continuous …

Ecoprime Twin – Scale-Up Of Capturesmb To The Process Scale, Kathleen Mihlbachler, Thomas Muller-Spath, Lars Auman, Michael Bavand, Rick Langone, Gerard Gach

Integrated Continuous Biomanufacturing II

Affinity chromatography is a powerful purification tool in pharmaceutical industry for capturing therapeutic proteins, in particular monoclonal antibodies (mAbs) and mAb fragments, from cell culture harvest. During the last years the resin utilization and productivity of affinity chromatography significantly improved due to the newly introduced multi-column continuous processes. The process setup was optimized to a twin-column continuous capture technology, known as CaptureSMB which requires less equipment hardware, has a less overall complexity and performs comparable if not better than other multi-column setups.

This presentation outlines the CaptureSMB concept and focuses on the scale-up from bench-top to twin-column pilot scale. Especially, …

Evaluating Facility Design And Capacity Planning Decisions For Clinical And Commercial Supply With Hybrid Continuous Processes, Suzanne Farid

Integrated Continuous Biomanufacturing II

Assessing the value potential of alternative process and facility design strategies will be critical to the successful management of continuous processes. UCL’s Decisional Tools team have developed advanced decision-support tools that effectively integrate concepts from bioprocess economics, dynamic simulation, risk analysis, combinatorial optimization and operations research to address such challenges (e.g. 1-5). This presentation will show our latest practical applications of such models to industrially-relevant problems related to facility design and capacity sourcing for long-term commercial supply when dealing with processes with either continuous upstream or downstream processes. The results can be translated across a range of products such as …

What Is Holding The Industry Back From Implementing Cbp (Continuous Bioprocessing) More Broadly?, Morten Munk

Integrated Continuous Biomanufacturing II

Introduction: Advances in pharmaceutical manufacturing technology in the last decade provide new opportunities to reinvigorate and improve the quality of pharmaceutical manufacturing. One of those technologies is Continuous Processing, which offers a wide range of advantages. Traditionally, most focus has centred on the cost advantages while the industry has held back on more broadly implementing Continuous Process. One reason for this reluctance concerns the viewpoint on the technology from the authorities, particularly FDA and EMA.

This presentation will discuss the results from a survey of industry peers performed by BioPlan Associates for NNE Pharmaplan. The focus of the survey is …

Novel Compact Cell Settlers For Continuous Perfusion Bioreactor Cultures Of Microbial (And Mammalian) Cells, Dhinakar Kompala, Brian Batt

Integrated Continuous Biomanufacturing II

About 25 years ago, we first demonstrated the use of inclined settlers for selective removal of dead cells and cell debris and complete recycle of live and productive cells back to perfusion bioreactors with murine hybridoma cells (Batt et al., 1990) and Chinese hamster ovary cells (Searles et al., 1994). These devices have been scaled up as lamellar settlers (Probstein and Yung, 1979) and applied successfully over the last two decades for commercial manufacture of several therapeutic biologics in high cell density continuous perfusion bioreactor cultures of recombinant mammalian cells by different biotech companies. However, the rectilinear scale up of …

Tools For Process Intensification Upstream And Continuous Processing Downstream, James Rusche

Integrated Continuous Biomanufacturing II

The intensification of cell culture production and continuous downstream processing are two important features of many new biomanufacturing schemes. Perfusion of cell culture bioreactors using an alternating tangential flow (ATF) device can increase viable cell density 5-10 fold and can be used to continuously harvest product suitable for immediate downstream processing. Perfusion can also be applied to high density cell banking and preparation of N-1 cell inoculum to greatly accelerate production turnover in fed batch processes. The capture of continuous upstream production can be made practical by prepacked columns of uniform performance and rapid load using low aspect ratio columns. …

Delivering Steady-State Product Quality With An Intensified And Integrated Perfusion Cell Culture Process, Jason Walther, Neha Shah, Myles Hollenbach, Jonathan Wang, Marcela Yu, Jiuyi Lu, Konstantin Konstantinoc, Chris Hwang

Integrated Continuous Biomanufacturing II

Continuous biomanufacturing provides many important strategic advantages for the production of protein therapeutics through process integration, simplification and intensification. To achieve upstream process intensification, Sanofi is currently developing robust cell culture processes that can achieve ultra-high cell densities and productivities (“push to high”) while minimizing cell-specific perfusion rates (“push to low”). We have applied ATF perfusion technology and improved the cell culture environment to achieve high cell densities and volumetric productivities with minimal ATF filter fouling. Meanwhile, we have employed high-throughput screening strategies to increase medium depth and reduce medium requirements. We will describe results as well as ongoing efforts …

Inline Spiking For Viral Clearance Validation Of Continuous Processes, Herb Lutz

Integrated Continuous Biomanufacturing II

Conventional clearance validation employs batch spiking using a uniform feed. In continuous processing, the feed to a unit operation can vary over the course of processing. An example would be an elution peak from an upstream step where the salt and protein concentrations vary over the course of operation. Batch spiking would not represent manufacturing practice in this case. Employment of inline spiking allows a scale down version of changing feed to be used for spiking studies. This talk will describe the qualification and application of the inline spiking method with a virus filter.

Enabling Technologies For Integrated / Continuous Downstream Processing Of Biologics, Jeff Salm, Marcus Fiadeiro, Raquel Orozco, Jill Kublbeck, Aaron Noyes, Jeff Horne, Daniel Lacasse, Ashley Sacramo, Suhani Gupta, John Coffman, Robert Fahrner

Integrated Continuous Biomanufacturing II

Pfizer Bioprocessing R&D is focused on developing enabling technologies that will reduce capital and operational expenses, decrease equipment scale, increase automation and utilize fewer FTEs.

To realize this vision, Purification Process Development has piloted new technologies and operational strategies that have enabled a fully integrated downstream process. Our current work has demonstrated a continuous process that includes tangential flow filtration harvest from a perfusion bioreactor, Protein A capture, inline viral inactivation/conditioning and AEX polishing. This process was fully automated and demonstrated at the 100 L scale. We have also shown feasibility of multi-day virus reduction filter operation and a continuous …

Case Study: Optimisation Of A Stabilised Large Scale Atf Perfusion Process, Jarno Robin

Integrated Continuous Biomanufacturing II

Mammalian cell cultures are used to produce a range of recombinant therapeutic proteins where post-translational modifications, such as glycosylation, are of key importance. The demand for proteins expressed in mammalian cells has increased rapidly over the last decade, primarily driven by the success of monoclonal antibodies (mAbs) that are required at large doses for therapeutic use. Several host cell lines are employed for manufacturing (e.g. NS0, hybridomas, PER.C6, BHK, etc.) but the primary workhorses are Chinese Hamster Ovary (CHO)-derived cell lines accounting for over 70% of the recombinant therapeutics. CHO cells have a proven track record as a safe host …

Efficient Approaches For Perfusion Medium Development, Thomas Falkman, Andreas Castan, Eric Faldt, Teres Persson, Jill Simon, Helena Nordvarg

Integrated Continuous Biomanufacturing II

Here, we present a fast and convenient strategy for developing a high-cell density perfusion process for antibody-producing Chinese hamster ovary (CHO) cells based on the commercially available ActiCHO™ Media System. ActiCHO P base medium was used as a starting point and ActiCHO Feed-A and Feed-B were added in various concentrations as supplements. The resulting perfusion medium prototypes were first evaluated in batch cultures, applying a design of experiment (DoE) strategy (Figure 1), and then tested in small-scale perfusion cultures in rocking single-use WAVE bioreactor™ systems (Figure 2). The medium optimization resulted in a final process with a cell-specific perfusion rate …

Design Criteria And Requirements For Development Of Perfusion Media, Jochen Sieck

Integrated Continuous Biomanufacturing II

There is increasing interest in perfusion processing as a means for increasing overall yield of the upstream process, facilitating a reduction of manufacturing costs. However, as in many companies the focus was on fed-batch process development for decades, hands-on knowledge about medium development for perfusion processes is considered a key gap for the wider adoption of perfusion processing in biologics manufacturing today.

Compared to batch-based cultivation, perfusion processes have fundamentally different requirements and challenges in terms of medium composition. The continuous nature of perfusion processes allows to achieve steady-state, although not all practitioners consider this desirable. The main challenge in …