Engineering Commons^™

Advances In Bioreactor Scale-Down Modeling Using Process Analytical Technology, Liying Yang, Jimmy Liu, Benjamin D'Alessio

Cell Culture Engineering XV

A bioreactor scale-down model is an essential element in process development and commercialization. In this poster presentation, key strategies in bioreactor scale-down model development and characterization will be discussed. A 4-Liter bioreactor scale-down model was developed to emulate a 15,000-Liter commercial scale bioreactor. Mass transfer characterization studies were conducted for the 15,000-Liter bioreactor, from which the results were summarized and utilized to guide scale-down model development / characterization. Case studies are shown where scale-down model representativeness is enhanced by implementing PAT. Specifically, automated dissolved carbon dioxide (pCO₂) control was implemented during scale-down model development in order to establish …

Single-Step Flask To 250 L Cell Culture With A Hybrid Mixing Single-Use Bioreactor, Nephi Jones, Tony Hsiao

Cell Culture Engineering XV

The process of scaling suspended cell cultures from frozen stocks through a large single-use bioreactor (SUB) includes numerous processing steps and operations. These steps, or seed train, involve vessel transfers and associated connections that increase the potential for contamination and human error. Of the multiple methods available for inoculating a 250 L SUB with sufficient cell quanitites, including multiple shake flasks, tabletop stirred tank bioreactor, or a 50L SUBs. Over the past decade tilting-type single-use bioreactor has been particularly attractive. Such wave-inducing systems implement tilting or rocking motion to agitate cultures which provides gas transfer and maintains cell suspension. Additionally, …

Scale Down Model In Industrial Cell Culture Processes – A Powerful Tool To Ensure Reliable Production, Marco Jenzsch

Cell Culture Engineering XV

Reliability and reproducibility of therapeutic protein production is one of the key objectives of GMP manufacturing with respect to product quality and supply to patients.

But even in the age of chemical defined media, QbD and well characterized cell culture processes during manufacturing deviations can occur. While those deviations caused by human errors, equipment failures or process control (e.g. pH) can be assessed in a straightforward manner coming to satisfying results in an appropriate period of time, deviation caused by raw material lot variations are usually hard to detect. Development of risk mitigation strategies is complex since sophisticated analytical methods …

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, …

A Holistic Approach To The Scale-Up Of A Microcarrier-Based Perfusion Cell Culture Process For The Production Of A Therapeutic Enzyme, Jin Yin

Cell Culture Engineering XV

This case study describes our holistic approach of scaling up a microcarrier-based perfusion cell culture process for the production of a therapeutic enzyme directly from a 12L benchtop glass bioreactor to a commercial scale stainless steel bioreactor. Besides conventional scale-up challenges such as mixing, shear stress and mass transfer, the scale-up of this microcarrier-based cell culture process posed its own challenges, e.g. the original design of the commercial scale stainless steel bioreactor had difficulty suspending microcarriers. Additional challenges included achieving effective cell attachment after seeding, balancing microcarrier stratification and culture turbulence, and minimizing bubble shear stress and foaming. To mitigate …

Characterization Of Tap Ambr250 Disposable Bioreactors As A Reliable Scale-Down Model For Biologics Process Development, Ping Xu, Colleen Clark, Todd Ryder, Charo Scott

Cell Culture Engineering XV

In recent years, biologic drugs such as antibodies, biosimilars and fusion proteins have continued to enter into the pharmaceutical pipeline. To shorten cell culture process development and accelerate clinical trials and regulatory filing, the disposable Advanced Microscale Bioreactors (Ambr 250) has burgeoned to be a useful tool due to the advantages of highly automated control and short turnaround. However, the traditional early stage upstream process development is normally conducted in 3L or 5L bioreactors. The usage of Ambr 250 instead of 5L bioreactors in the development has many benefits, but also brings necessity to establish a scale-down model of Ambr …

Scale-Up In The Single Use Age: Does Geometry Matter?, Colin Jaques, Anthony Beaney

Cell Culture Engineering XV

Singe use bioreactors (SUBs) are becoming standard work horses in the biopharmaceutical industry. These SUBs are supplied by vendors as off the shelf designs limiting the cell culture engineer’s ability to match the geometry of the SUB to the geometry of their existing stirred tank reactor (STR) capacity. The first generation of SUBs departed from conventional stirred tank bioreactor (STR) geometry in terms of impeller number, and orientation and sparger hole diameter. Moreover, one marked feature of SUB bioreactors was that they could be operated at lower volumes than conventional STRs, bringing considerable operational flexibility. This practice, however, further negated …

A Rapid Approach For Basal And Feed Media Optimization In Ambr® 15 Bioreactors, Michael Gillmeister

Cell Culture Engineering XV

A Chemically-Defined (CD) and Animal Origin-Free (AOF) custom media optimization approach was developed using the ambr^® 15 microbioreactor system. This approach combines pre-qualified blends of well-characterized media, a series of high-throughput Design of Experiment (DoE) studies and analyses to optimize a fed-batch process, and on-site dedicated media application specialist support. This system can be utilized when commercially available media fall short of performance expectations and high-producing cell lines must be developed as quickly as possible. In comparison, fully customized media and feed development requires significant funding, time, and in-house expertise in media development. Using this media optimization approach, a …

Process Scale-Up Issues: Relics Of The Past Or Continues To Cause Major Headaches, Sadettin Ozturk

Cell Culture Engineering XV

Since the inception of biopharmaceutical industry, scale up issues hampered the success of process transfers and product commercialization. In the last 30 years, the field evolved significantly, we have gained significance experience, broadened our understanding of cell culture, and resolved issues such as shear, mixing, and aeration. Requirement of maintaining geometric similarity and scale-up parameters (power per volume or tip speed) was proven not to impact the performance of the bioreactors significantly. Today’s processes could be scaled up from micro-bioreactors to full-scale production bioreactors rather easily. This talk will discuss the factors that played a significant role for this accomplishment. …

Novel, Efficient Scale-Up Of Inclined Settlers For Perfusion Bioreactor Cultures, Dhinakar Kompala

Cell Culture Engineering XV

Inclined settler has been introduced as a unique cell retention device for perfusion bioreactor cultures over 25 years ago by these authors (Batt et al, 1990; Searles et al. 1994) to selectively remove dead cells and cell debris, while the live and productive cells are recycled back to bioreactor. This device has been scaled up successfully as lamellar settlers and utilized to achieve high cell densities and productivities in production-scale perfusion bioreactors by a number of biotechnology manufacturers, such as Bayer, Biomarin, Eli Lilly, Roche, etc. (Shen and Yanagimachi, 2011; Pohlscheidt, et al., 2013). However, the rectilinear scale up of …

Scale-Up And Scale-Down Topics Facing The Industry, Markus Michael Muller

Cell Culture Engineering XV

Scale-up and scale-down is bringing several challenges along. We present some recent examples where these challenges occur and how extensive hardware characterization by for example computational fluid dynamics has leads to get a better understanding of the processes themselves.

The increasing implementation of small scale bioreactor systems like ambr15 and ambr250 as well as the usage of new single use bioreactors (>2-L) in parallel to established glass vessels has led to additional challenges in scale-up and scale-down. The mini-bioreactor systems, originally used for screening approaches, are going to be implemented as small scale testing systems but could also be …

Case Study For Improved Process Robustness At Manufacturing Scale For A Mammalian Cell Culture Process: Troubleshooting Medium Preparation And Gas Entrance Velocity Effects, Robin Luo

Cell Culture Engineering XV

A case study is presented of the scale up of a mammalian cell culture process from 100 L to 2000 L scale. Upon scale up, decreased culture growth and productivity were observed, along with batch to batch variation. The reduced performance at 2000 L scale was attributed to two factors: early culture acidification, resulting from differences in medium preparation, and higher gas entrance velocity. Troubleshooting was performed at bench, pilot, and manufacturing scale for developing improved process understanding. The medium preparation time and the associated base titration were shown to be important factors impacting process performance. In addition, with the …

Establishing A Ph Measurement Reference Method For Site/Process Transfer Purposes, Meg Tung, Jacob Mauger, Christian Klinger, Detlef Eisenkratzer

Cell Culture Engineering XV

pH is an important cell culture process parameter that must be carefully monitored and controlled to ensure optimal culture growth, productivity, and product quality. In mammalian cell culture processes, online bioreactor pH sensors are periodically recalibrated against an offline pH value generated by a reference instrument to allow true culture pH to be maintained at or near the target pH setpoint.

Within the Roche R&D and manufacturing network a variety of instrumentation are used to obtain offline pH measurements. While internal guidance exists for pH monitoring, differences in offline pH measurements – due to site-specific instrumentation and procedural differences – …

Preferentially Selecting Cellular Metabolism And Improving Productivity By Controlling Do And Pco2, Jiayi Zhang, Ryan Cassidy, Mark Emanuele, Gonzalo Milet, Lada Laenen

Cell Culture Engineering XV

Cells utilize glucose as their main resource for deriving energy through ATP production. The quantity of ATP generated depends on the metabolic pathways that are employed, aerobic glucose metabolism or anaerobic glucose metabolism. Using our bench top bioreactor model, we have shown these two metabolic pathways can be preferentially selected by controlling the desired cell culture DO and pCO2, and productivity was increased as a result. The DO and pCO2 controlling strategy was implemented in at-scale bioreactors and yielded the expected metabolic and productivity outcome

Optimization Of Glycosylation And Charge Distribution Through Culture Parameters And Supplements, Sigma Mostafa, Ventaka Tayi, Shahid Rameez, Nathan Oien, Jaspreet Notey, Brian Baker, Jimmy Smedley, Abhinav Shukla

Cell Culture Engineering XV

Culture parameters are known to have significant impacts on product quality, although these effects are sometimes cell-line dependent and the directionality of the effect has to be determined empirically. We will present data from three case studies where glycosylation or charge distribution was modified to match the reference molecule or to reduce variability. In the first case study, glycan optimization for a biosimilar will be described. Galactose, fucose, and mannose levels were optimized through screening of raw materials and process parameters. A range of media, feed, alternate sugars, metals and other supplements as well as temperature set points were tested. …

From Observation To Control: Using Cell Culture Automation For Enhanced Product Quality Optimization, Sven Markert

Cell Culture Engineering XV

In this talk we will present an overview of our findings on influencing and controlling product quality attributes of different antibody derived biopharmaceuticals like standard IgGs, bispecifics, glycoengineered and antibody cytokine fusion proteins.

We used the Roche in-house developed automated cell culture system which is based on shaken multi-well plates and a fully automated process workflow. More than 600 fed-batch cultivations can be handled in parallel enabling the screening of a wide range of process conditions. Combining the automation technologies in the fields of cell culture and product quality analytics enables the high throughput screening of a broad range of …

3d Tumor Models With Defined Cellular And Physico- Chemical Components: Impact Of Recapitulative Tumor Microenvironment On Disease Progression, Catarina Brito, Vitor Santo, Marta Estrada, Sofia Rebelo, Elizabeth Anderson, Wolfgang Sommergruber, Erwin Boghaert, Paula Alves

Cell Culture Engineering XV

The high attrition rates observed in cancer drug discovery (up to 95% of failure of drugs tested in phase I trials) have raised the awareness of the scientific and industrial communities towards the need for more predictive pre-clinical models. These models should be more representative of the disease and consequently help to eliminate at pre-clinical stages drug candidates that lack efficacy or safety. Tumor microenvironment is composed by a network of fibroblasts, endothelial cells, immune-competent cells within the extracellular matrix (ECM). Interactions between these components are critical for tumor initiation, proliferation, migration and metastasis. The design of in vitro cancer …

Performance Consistency Of Fed-Batch Cultures Across Multiple Systems Used In Upstream Process Development, Matthieu Stettler, Jean Marc Bielser, Yolande Rouiller, Martin Jordan, Herve Broly

Cell Culture Engineering XV

Each stage of cell culture process development requires fit for purpose tools. The selection of a fed-batch cultivation system is often based on throughput and cost. However, the process knowledge derived from different systems and scales is not necessarily identical. Hence, a careful evaluation of systems which are already established or newly implemented is essential. We recently introduced a novel high throughput fed-batch screening system (1) and the objective of this study was to provide data on how it compares with other systems used in early and late stage cell culture process development. We describe the performance of 12 different …

Challenges And Their Resolutions During Process Development And Tech Transfer Of A Late Stage Bispecific Antibody Product, Marcela Yu

Cell Culture Engineering XV

During the Phase III process development and tech transfer of a bispecific antibody project in Sanofi, many technical and logistical challenges were encountered for this cross-site collaboration project. In particular, Phase III process development and tech transfer involves three different sites as sending and receiving units located in different continents using different scales of equipment. Due to this constraint, additional considerations are required to address gaps between different small scale models prior to scale-up in order to de-risk the challenges that we may encounter during process transfer. Particularly, computational fluid dynamics (CFD) modeling and first principles calculation are used not …

Cell Culture Engineering And Biosimilars: The Physician’S Perspective, Jan Hillson

Cell Culture Engineering XV

Biosimilars promise to increase access to life-saving and life-enhancing therapeutics, while creating cost savings that can fund further innovations in health care. Achievement of these goals is critically dependent on acceptance by prescribing physicians. This session will explore the following questions: What do clinicians care about? What do they know about the manufacture and characterization of biosimilars? What should they know? These questions will be addressed in the context of specific examples arising in the development of biosimilars to Neupogen, Infliximab, Humira, and Rituximab.

Bioreactor Scale-Up Harmonization - From Process Development To Manufacturing, Claudia Berdugo-Davis

Cell Culture Engineering XV

Successful bioprocess scale up requires an understanding of the microenvironment influenced by bioreactor parametric conditions and the effect on cellular metabolism and expression of a recombinant protein. While, significant understanding of best practices exists in scaling up bioprocesses, Contract Development and Manufacturing Organizations (CDMO) have a unique challenge in requiring a working knowledge of our bioreactor capabilities driven by a diverse client network and thus a need for a ready-to-implement scale up platform for a wide range of molecule types. An effective CDMO must be able to scale-up client platform processes (which differ for each client) as well as utilize …

Tubespins As A Suitable Scale-Down Model Of 2l High Cell Density Bioreactors For Cho Cell Culture, Natalia Gomez

Cell Culture Engineering XV

High cell-density culture of Chinese Hamster Ovary (CHO) cells significantly increases cell growth, viability and volumetric productivity of recombinant therapeutic proteins. These improvements can reduce size or number of manufacturing cell culture vessels, decrease plant footprint and ultimately, the cost of goods.

Process development is typically performed in instrumented lab-scale bioreactors, which, while effective, require time and labor for set-up and operation. Is it attractive to develop alternate systems that provide representative results but without the effort of bioreactor operation. To this effect, we evaluated 50 mL Tubespins as a model to mimic high cell density cultures.

We tested 13 …

Integrated Continuous Bioprocessing - A Gold Mine For Cell Culture Process Understanding?, Mats Akesson, Martin Heitmann, Ditte Skibstrup, Peter Tiainen

Cell Culture Engineering XV

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]. 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 a 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 the …

Identification Of Cell Culture Levers To Lower Trisulfide Modifications In Monoclonal Antibodies Produced In Cho Cell Culture, Masaru Shiratori, Jessica Wuu, Martin Gawlitzek

Cell Culture Engineering XV

Trisulfide bond formations are a commonly observed modification of monoclonal antibodies produced in cell culture processes. Protein trisulfide bonds are post-translational modifications in which an extra sulfur atom is bound between two cysteines involved in disulfide bonding. This modification has been detected in both recombinant and endogenous plasma-derived IgGs. Inconsistent levels of trisulfides were identified as a source of variability observed during the conjugation process for antibody-drug conjugates (ADCs). To understand the root cause for variable trisulfide levels observed during process development for different molecule projects, cell culture studies aimed at identifying process levers to minimize trisulfide formation were conducted. …

Metabolomic Analysis For Scale-Down Model Improvement, Eric Garr

Cell Culture Engineering XV

The use of scale-down models is essential for the development, characterization and continuous improvement of commercial cell culture processes. A model demonstrated to be predictive of large-scale performance is advantageous as small-scale data can be used to support process investigations and changes, reduce costs and consequently accelerate new therapies to market. Aligning geometric aspect ratios and dimensionless engineering parameters improved the overall scale-down model representation for key process outputs such as pCO₂ accumulation and cell viability. Despite these changes, the revised scale-down model did not fully match the large-scale process lactate accumulation. Interestingly, it was demonstrated that pCO_{2 …}

Application Of Multivariate Data Analysis In The Monitoring And Control Of Mammalian Cell Processes, Stephen Goldrick, Richard Turner, Marcel Kuiper, Kenneth Lee, Rahul Pradhan, Suzanne Farid

Cell Culture Engineering XV

High throughput (HT) methodologies are increasingly being adopted for bioprocess development activities. However, often the large quantities of data generated from such studies as well as from historical batch records are not fully harnessed for their potential insights. Multivariate data analysis (MVDA) is a well-known technique that can reduce the dimensionality of large data sets and help generate useful correlations of typical process behaviour and determine root causes of process deviations.

This work focuses on the application of various MVDA techniques and data mining tools to investigate the impact of process variations on the critical quality attributes (CQAs) of mammalian …

Evaluation Of Public Genome References For Rna-Seq Data Analysis In Chinese Hamster Ovary Cells, Huong Le, Chun Chen, Chetan Goudar

Cell Culture Engineering XV

Recent advances in next-generation sequencing technologies have led to the emergence of RNA-Seq as the preferred transcriptomic tool in the biopharmaceutical industry. However, an important challenge with deploying RNA-Seq to characterize CHO cells is the absence of a common genomic reference for this species. In most published CHO cell transcriptomic studies, RNA-Seq reads are assembled into de novo genomic references which were subsequently used for mapping of the constituent reads. Such an approach makes it difficult to compare results across studies due to the incomplete and non-universal nature of those assemblies. To address this challenge, we evaluated two publicly available …

Application Of Online Co2 Monitoring To Enable A Better Understanding Of Cell Culture Performance Variation Between Gmp-Scale And Scaled-Down Bioreactors, Ting Kuo Huang, Jacob Mauger, James Dvornicky

Cell Culture Engineering XV

Dissolved carbon dioxide (dCO₂) or partial pressure of carbon dioxide (pCO₂) is an important process parameter that may impact process performance (product titer and viability) and product quality attributes (such as glycosylation) during mammalian cell culture process in a bioreactor. The impact of altered level of pCO₂ on the cell culture process may manifest itself upon process scale-up if the CO₂ removal rate is not consistent between the development scale and the manufacturing scale. The pCO₂ level during cell culture process is normally determined through offline measurement; however, the offset between online and …

Cardiac Remodeling And Dysfunction In Childhood Obesity: A Cardiovascular Magnetic Resonance Study, Linyuan Jing, Cassi M. Binkley, Jonathan D. Suever, Nivedita Umasankar, Christopher M. Haggerty, Jennifer Rich, Christopher D. Nevius, Gregory J. Wehner, Sean M. Hamlet, David K. Powell, Aurelia Radulescu, H. Lester Kirchner, Frederick H. Epstein, Brandon K. Fornwalt

Pediatrics Faculty Publications

Background: Obesity affects nearly one in five children and is associated with increased risk of premature death. Obesity-related heart disease contributes to premature death. We aimed to use cardiovascular magnetic resonance (CMR) to comprehensively characterize the changes in cardiac geometry and function in obese children.

Methods and results: Forty-one obese/overweight (age 12 ± 3 years, 56 % female) and 29 healthy weight children (age 14 ± 3 years, 41 % female) underwent CMR, including both standard cine imaging and displacement encoded imaging, for a complete assessment of left ventricular (LV) structure and function. After adjusting for age, LV mass index …

Highly Conductive In-Sno2/Rgo Nano-Heterostructures With Improved Lithium-Ion Battery Performance, Ying Liu, Alessandro Palmieri, Junkai He, Yongtao Meng, Nicole Beauregard, Steven L. Suib, William E. Mustain

Faculty Publications

The increasing demand of emerging technologies for high energy density electrochemical storage has led many researchers to look for alternative anode materials to graphite. The most promising conversion and alloying materials do not yet possess acceptable cycle life or rate capability. In this work, we use tin oxide, SnO2, as a representative anode material to explore the influence of graphene incorporation and In-doping to increase the electronic conductivity and concomitantly improve capacity retention and cycle life. It was found that the incorporation of In into SnO2 reduces the charge transfer resistance during cycling, prolonging life. It is also hypothesized that …