Engineering Commons^™

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 …

Considerations For An Incubation Chamber For Continuous Viral Inactivation, Raquel Orozco, Nancy Guillen, Loleta Chung, Scott Godfrey, Jon Coffman

Integrated Continuous Biomanufacturing II

Continuous bioprocessing offers numerous benefits that have been highlighted recently in the literature. While substantial research efforts have allowed for relevant developments in continuous cell culture processes, downstream challenges, such as continuous viral inactivation, have not been addressed. The purpose of this work is to design a chamber that incubates a continuous product stream for a desired incubation time, typically for 1 hour. Since plug-flow cannot be achieved at typical incubation times and flow rates, one of the biggest challenges is to address dispersion of the product stream. Since several logs of viral clearance should be achieved during a virus …

Optimal Control Of A Continuous Bioreactor For Maximized Beta-Carotene Production, Nazmul Karim, Jonathan Raftery, Xinghua Pan

Integrated Continuous Biomanufacturing II

Carotenes such as β-carotene have a positive impact in human health as a precursor of vitamin A. Structural complexity of these compounds makes their chemical synthesis a difficult endeavor, facilitating the need for their biological production. Continuous operation can be used in these processes to allow for increased cell viability through the mitigation of substrate and product inhibition, thereby increasing the productivity of the carotenoid product. This work looks to examine the operating conditions necessary to achieve the maximum production of the desired carotenoid products when produced via continuous operation. First, a two-level parameter estimation method is utilized to develop …

Process Time And Cost Savings Achieved Through Automation And Islands Of Integration In Existing Facilities., Rob Noel, Yuki Abe, Andrew Sinclair, Lynne Frick

Integrated Continuous Biomanufacturing II

For most biopharmaceutical manufacturers the cost, time and quality improvements offered by adopting continuous manufacturing operations is, as with any new technology, inhibited by the research and validation costs associated with early adoption. Additional effort is needed to validate the novel technology in-house. There are simple changes that can be made in disposable biomanufacturing , which are necessary for the eventual adoption of full continuous operations, and yet have benefits now. These step changes are shown by economic & scheduling models to offer direct cost savings and improved facility throughput. A road map of these step-wise improvements is provided that …

Spectral Deconvolution Of Chromatograms Without Offline Analytics, Matthias Rudt, Nina Brestrich, Florian Dismer, Jurgen Hubbuch

Integrated Continuous Biomanufacturing II

Continuous bioprocessing has shifted into the focus of the pharmaceutical industry in recent past. An important driving force is the expected higher productivity compared to operations in batch mode. To ensure constant quality attributes over a complete production cycle, reliable process analytical technologies (PAT) are central.

The deconvolution of spectral measurements in downstream processing has been introduced previously as a powerful PAT tool[1]. For spectral deconvolution, published research focused on partial least square (PLS) regression models. PLS models are able to deconvolute spectra even if the spectral differences of proteins are minute. They are widely used in chemometrics. However, PLS …

Genomics Based Methodology Of Cell-Culture Media Formulation For Improved Biotherapeutic Productivity And Quality Consistency, Hemlata Bhatia, Seongkyu Yoon

Integrated Continuous Biomanufacturing II

Biosimilar drugs are emerging very fast as the patents of innovator’s drugs are expiring. Media formulation development has to be carried out for each bio similar, which takes up a significant amount of time and generally, a random approach is taken to meet comparability requirement and improve the product titer. A targeted approach with genome information of host cell as a function of media composition can provide useful information to explain the product titer variability and comparability. A 2-step correlation model explaining relationship between cell-culture productivity and quality, and media compositions taking gene expression as intermediate attributes can provide a …

Simple Method Transfer From Batch To Continuous Chromatography Process To Fit Parameters To Business Needs, Rene Gantier, Karl Rogler, Xhorxhi Gjoka, Ron Noel, Alexander Martino, Mark Schofield

Integrated Continuous Biomanufacturing II

Multicolumn chromatography (MCC) is a proven technique to significantly improve performance of bio-processes by increasing specific productivity, reducing volume of resin used and enabling disposable, continuous and integrated processes. But the transfer of process parameters from batch to continuous mode is perceived as complex and could limit adoption at clinical and manufacturing scales.

We have developed and tested a new method to translate data from a minimal number of single column runs into a multicolumn process with predictable performance at large scale. Depending on the titre of the product and the processing time required, the method predicts the optimal number …

Integrated And Single Use Continuous Manufacturing, Atul Mohindra

Integrated Continuous Biomanufacturing II

Continuous processing for biopharmaceutical production is attracting considerable interest within the industry. The concept of continuous processing is not new and the value of continuous manufacturing has been well documented for decades across the steel, chemicals and food industries. Despite the vast differences between product types, the advantages of continuous over batch production remain consistent and include steady state operation, high volumetric productivity, streamlined process flow and reduced capital costs.

Over the past decade, significant investments have already been made with continuous bio-manufacturing development. The need for the biopharmaceutical industry to follow suit is being influenced by a number of …

Pilot Scale Hybrid Fed Batch And Continuous Processing Of Biologics, Dave Sullivan, Michael O'Connor, Samir Gondalia, Matt Gagnon

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, our Pilot Facility has partnered with our cell culture process development colleagues to adapt a fed batch platform 150L stainless steel bioreactor to run in hybrid perfusion, standard perfusion, low volume cell controlled perfusion, and continuous stirred tank modes. Through adjustments to impeller configuration, sparging strategy, and addition ports the bioreactor was able to deliver multiple batches that produced ~3X gains in cell density and volumetric productivity versus conventional fed batch …

Comparison Of Bioreactor Systems Operated At High Bacterial Cell Density For The Production Of Lactic Acid: Batch – Cstr – Cstr Cascade – Tubular Reactor, Ulrich Kulozik

Integrated Continuous Biomanufacturing II

Subject of the work was the microbial conversion of lactose, an abundant processing by-product in the dairy industry, to lactic acid. Lactic acid serves as a preservative in many areas in various product sectors, but its potential applications go far beyond this. In industrial applications lactic acid as a renewable material can be applied as a building block for novel technical materials such as biodegradable films for packaging purposes in replacement of materials based on fossile raw materials.

Purpose of the work was to compare different bioreactor systems with regard to achievable lactic acid concentrations and volumetric productivities. The goal …

Continuous Downstream Process Or Connected Batch Process: Which One Makes Most Sense For Biogen?, John Pieracci, Sanchayita Ghose, Venkatesh Natarajan

Integrated Continuous Biomanufacturing II

As biologics-based products move into therapeutic areas with large patient populations and high doses, batch processing may not be able to keep pace with product demands. At Biogen, we have been exploring a number of options that can enable higher productivity of our downstream processes. In addition to a fully “end to end” continuous process, a batch process comprised of several steps connected in series has been evaluated. In this presentation, technologies Biogen has evaluated to enable either continuous or connected processing will be shown. Multi-Column-Chromatography (MCC) for the Protein A capture chromatography step was evaluated in order to maximize …

Continuous Culture In The Age Of Single-Use, Christel Fenge, Melisa Carpio

Integrated Continuous Biomanufacturing II

Continuous culture offers a number of advantages, e.g. higher titres and constant culture environment and is the method of choice for challenging therapeutic proteins such as coagulation factors. Additional opportunities arise for the commercial production of monoclonal antibodies using so called concentrated fed-batch mode where the product is retained in the bioreactor and fresh medium is constantly perfused through the culture and combining it with single use production technologies. This approach is an effective way to reduce time to market as additional scale-up steps after phase III are not required to produce large amounts of product whilst benefiting from the …

Pat Concepts For The Process Monitoring And Control Of Continuous Biomanufacturing, Eike Zimmerman, Jeff Goby, Anoushka Durve

Integrated Continuous Biomanufacturing II

Process Analytical technologies (PAT) are a key component to fully leverage the success of continuous manufacturing. The timely measurement of critical quality attributes and critical process parameters assures that the desired product quality is being consistently manufactured and non-conforming material being identified. PAT tools can be utilized to meet the expectations for in-process monitoring, and allows for identification and isolation of rejected in-process materials. It further supports process development, as continuous and fast response of process to factor changes allows efficient experimentation and increases process understanding within the range of conditions studied during development. Ultimately, PAT will allow Real Time …

Upstream Perfusion Process: Back To The Future, Jean-Marc Bielser, Henri Kornmann, Herve Broly

Integrated Continuous Biomanufacturing II

At an early stage of bioprocess science, continuous operations were the workhorse in the industry. Therefore, EMD-Serono has legacy with continuous upstream processes as many of its molecules such as Rebif are being produced in perfusion mode. These processes from the 90’s consume large volumes of commercial media and demonstrate low productivities. Cells are mainly attached because no performant cell retention device existed for cells in suspension at that time.

Around 10-15 years ago, the industry decided to move on fed-batch operations. EMD-Serono also re-oriented towards fed-batch operations. Molecules such as Erbitux are now being produced in 15,000 L stainless …

Development Of Functionally Closed Downstream Operations For Continuous Biomanufacturing Of Recombinant Therapeutic Proteins, Rohan Patil, Rahul Godawat, Tarl Vetter, Yogesh Waghmare, Konstantin Konstantinov, Veena Warikoo

Integrated Continuous Biomanufacturing II

Traditionally, upstream processing in biologics manufacturing is performed in an aseptic and functionally closed manner while downstream processes are open operations designed to maintain a low-bioburden state. Consequently, the downstream process components such as skids, column hardware, and chromatography resins currently available have been designed for low bioburden rather than aseptic operations. One of the key design elements of Integrated Continuous Biomanufacturing (ICB) is the linkage of upstream and downstream operations and the capability of the system to run for prolonged durations at ambient temperature. Thus, a critical design criterion for ICB is the ability of the system to maintain …

Continuous Production Of Viral Vaccines With A Two-Stage Bioreactor System, Felipe Tapia, Yvonee Genzel, Ingo Jordan, Volker Sandig, Udo Reichl

Integrated Continuous Biomanufacturing II

Continuous processes can be particularly efficient for production of biologicals that are required in large amounts such as viral vaccines. One virus that has received much clinical attention is Modified Vaccinia Ankara virus (MVA), which is a potential platform for expression of recombinant viral antigens and can be used as a vector in gene therapy [1]. Recently, a new MVA virus strain has been successfully propagated at high yields in non-aggregated avian suspension cells [2] allowing the production of MVA virus in continuous bioreactors. MVA is a lytic DNA virus and therefore, continuous production strategies can be implemented using two-stage …

Bioprocess Economics And Optimization Of Continuous And Pre-Packed Disposable Chromatography, Richard Allmendinger, Suzanne Farid

Integrated Continuous Biomanufacturing II

The biotech sector is facing increasing pressures to design more cost-efficient, robust and flexible manufacturing processes. Standard batch chromatography (BATCH) is an established but expensive approach to separate impurities related with both E.coli and mammalian cells expression systems. This study uses a computational framework to investigate if the application of continuous chromatography (CONTI) and disposable technologies can provide a competitive alternative to BATCH and reusable equipment. A set of general assumptions is presented on how some of the key downstream processing characteristics, such as chromatography operating conditions, resin properties and equipment requirements, vary as a function of the chromatography mode …

Mathematical Modeling Of A Bioreactor Producing Epo-Hr Operating In Perfusion Mode, Osman Fernandez, Raydel Alvarez, Ernesto Chico, Adolfo Castillo, Julio Dustet

Integrated Continuous Biomanufacturing II

The interest in use perfusion mode has increased in the last years, due to an increased awareness of perfusion advantages, some general improvement in equipment reliability, and a broadening of operational skills in the biomanufacturing industry. However, mathematical modeling of bioreactors in continuous mode with cell retention (perfusion mode) with concomitant cell bleeding is still emerging, because this scheme has a few applications in global biotechnology industry. The case of study was industrial fermentation process of CHO cells producing EPO-hr. The model involves a total of 7 equations and 19 variables. In order to fix the degrees of freedom were …

Toward Complete Continuity In Antibody Biomanufacture: Multi-Column Continuous Chromatography For Protein A Capture And Mixed Mode Hydroxyapatite Polishing, Anthony Grabski, Tom Van Oosbree, Beth Hammer, Alla Zilberman, Robert Mierendoff

Integrated Continuous Biomanufacturing II

Monoclonal antibodies (mAbs) are a predominant modality for a broad range of clinical indications including oncology and inflammatory diseases. Increasing manufacturing capacity and decreasing cost per purification campaign are critical factors for making antibody therapies more affordable. Cell culture mAb expression levels have steadily increased over the past ten years with titers of five grams per liter frequently achieved. Higher titers reduce production costs and allow processing of kilogram quantities for clinical trials from single use cell culture vessels. Drawbacks of increased titers include higher levels of aggregates, fragments, variants, and process impurities. These combined titer and impurity burdens further …

Strategy For Scaling Semi-Continuous Downstream And Integration Of Process Analytical Tools For Monoclonal Antibody Toxicology, Darshini Shah, Rebecca Chmielowski, Colette Cutler, Hong Li, David Roush, Nihal Tugcu

Integrated Continuous Biomanufacturing II

Currently, continuous processing for biologics is being pursued from a technology perspective in order to increase productivity and decrease cost of manufacture. Disruptive technologies, such as PCC (Periodic Counter-current Chromatography), have been researched extensively in order to reduce the bottleneck for the downstream capture chromatography step. However, there is extremely limited knowledge in how to scale these disruptive technologies for clinical and commercial manufacturing. In this presentation, we demonstrate a strategy for scaling and integration of a PCC capture step into a semi-continuous downstream process (Protein A chromatography-Viral inactivation-Filtration-Anion exchange chromatography). Harvest cell culture fluid (HCCF) feed streams ranging from …

Biopharmaceutical Capacity Planning For Batch And Semi-Continuous Bioprocesses Under Various Strategic Criteria, Cyrus Siganporia, Thomas Daszkowski, Andreas Schluk, Brijesh Rao, Lazaros Papageorgiou, Suzanne Farid

Integrated Continuous Biomanufacturing II

Biopharmaceutical companies with expanding portfolios of commercial therapeutics face increasing pressure to meet market demands whilst minimising costs and capital expenditure. Attaining optimal production plans is made more problematic by portfolios containing products with different production modes: batch and semi-continuous. Semi-continuous perfusion-mode products often exhibit a distinct separation of upstream and downstream manufacturing via an intermediate freezing step. This flexibility adds further complexity which needs to be efficiently overcome during the optimisation process. An added complexity to having different process modes is that changeover times are different, leading to computationally expensive sequence-dependent changeover times. Considering the implications of incorrect capacity …

Development Of A First Generation Perfusion Process And Medium For Continuous Processing Based On Existing Fed-Batch Platform Media, Henry Lin, Samantha Wang, Robert Leighty, Scott Godfrey, Samet Yildirim, Adam Osborne, Flaka Radonigi, Jens Vogel, Jon Coffman

Integrated Continuous Biomanufacturing II

Process intensification leveraging perfusion offers tremendous potential for yield improvement over fed-batch processes for the production of monoclonal antibodies. In the context of continuous processing, the goal is to achieve highly intensified perfusion processes that allow substantial footprint reduction and enable flexible adaptation in new facilities. However developing a perfusion process and medium without prior technology requires leveraging the existing fed-batch platform knowledge. Evolving a medium for perfusion relies on designing suitable mixtures of basal and feed media that serve as adequate starting points for development. Focus on optimization of the medium to decrease byproduct waste, reduce unnecessary cell growth …

Scale Up And Implementation Of A High Density Long-Term Perfusion Suspension Cell Culture In A 250l Single Use Bioreactor, Weichang Zhou, Hang Zhou

Integrated Continuous Biomanufacturing II

As part of efforts to develop a continuous processing platform for biologics manufacturing using a single use bioreactor, we have been focusing on development of several initial unit operations: high density perfusion suspension cell cultures and early product capture steps, in order to realize its potentials of being flexible, improving product quality and lowering costs. These initial unit operations require large volumes and represent the most important part of this processing platform. By integrating these steps into a continuous operation, we can deliver the largest benefits of this processing platform. In this presentation, we will discuss our efforts towards continuous …

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 …