Biomedical Engineering and Bioengineering Commons^™

Poly-Pathway Model Approach: Simulation Of Multiple Metabolic States, Erika Hagrot, Hildur Oddsdottir, Anders Forsgren, Veronique Chotteau

Cell Culture Engineering XV

Animal cell lines have a complex and flexible metabolism and can display varied metabolic behavior depending on the culture conditions. A model that simulate and predict these variations would be a precious tool in the development of media, feeds or processes. However, in order to function as a predictive tool such a model has to describe the multiple metabolic states that can occur for a variety of conditions. This leads to the challenge of identifying a flexible model structure of relevant metabolic pathways and kinetics. To address this challenge, we have introduced the poly-pathway model approach aiming at capturing …

Systems Analysis Of Cho Cell Metabolism For Enhanced Fed-Batch Process Performance: Identification Of Novel Growth Inhibitors And Their Control, Bhanu Chandra, Jaitahree Kale, Anamaria Ovalle, Gregory Hiller

Cell Culture Engineering XV

Over the past decade, process development efforts have led to attaining titers close to 3-4g/L on a routine basis in CHO fed-batch cultures. However, with the advent of biosimilars and the ever expanding therapeutic portfolio of innovator drugs, there is a growing need for enhancing the throughput of fed-batch cultures in order to meet the product requirements with the limited manufacturing capacity. The classical hurdle for higher productivities in fed-batch cultures is the sub-optimal metabolism of mammalian cells that causes accumulation of byproducts, including lactate and ammonia, resulting in growth inhibition. With a robust CHO expression system, our group has …

The Potential Of Small Molecules To Modulate Glycosylation By Media Design, David Bruehlmann

Cell Culture Engineering XV

Background and novelty

A large number of recent publications demonstrate the effect of cell culture media on post-translational modifications of recombinant proteins¹. This study aims to extend the toolbox of media design beyond the commonly known media components. We identified and tested a large variety of cell culture compatible chemical components such as pigments and sugar derivatives in industrial relevant Chinese hamster ovary cell lines (CHO) expressing recombinant antibodies.

Experimental approach

The cells were cultivated in fed-batch mode cultures using shaking 96-deepwell plates, and process performance such as viable cell density, viability and product titer were monitored. Supernatants …

Case Study: Lessons Learned During Tech Transfer At A Multi-Product Legacy Launch Facility, Arthi Niarayanan

Cell Culture Engineering XV

Genentech's original GMP production facility in South San Francisco, California, was approved by the FDA in 1985 and today is licensed to manufacture 10 marketed products. This legacy manufacturing plant has evolved over the years to become a complex multi-product facility that supplies both clinical and commercial products utilizing a 96,000 liter-capacity on the CHO line. The site’s flexibility along with proximal location of key functional areas including Operations, Process Development, Quality and Regulatory well position it as a launch site for new molecules. Balancing flexibility with older qualified equipment presents unique opportunities when launching new molecules in an aging …

Factors Affecting The Sialylation Of Fc- Fusion Protein In Recombinant Cho Cell Culture, Gyun Min Lee

Cell Culture Engineering XV

Over the past decade, product titers from recombinant CHO cells have increased significantly. However, not only the product titer but also the product quality is important for therapeutic efficacy. When sodium butyrate (NaBu) was added to a culture of CHO cells producing Fc-fusion protein, it increased Fc-fusion protein titer, but it decreased sialylation of the Fc-fusion protein. Analysis of N-glycosylation-related genes revealed that altered expression patterns in st3gal3, neu 1, and neu3, which have roles in the sialic acid biosynthesis pathway, correlated with reduced sialic acid content of the Fc-fusion protein by NaBu. St3gal3, sialic transferase, was down-regulated and, nue1 …

Challenges Of Scale Down Model For Disposable Bioreactors: Case Studies On Growth & Product Quality Impacts, Jincai Li, Guangming Zhang, Huilin Zhu, Weichang Zhou

Cell Culture Engineering XV

Despite wide-spread use of disposable bioreactors, there is a lack of well-established scale-down model for larger scale SUBs. Here we report a case of NS0 cell culture process transfer from 2000L stainless steel bioreactor (SST) to 2000L disposable bioreactor (SUB). Initial attempts in trying to grow the NS0 cells in the small scale 2D bags yielded non-satisfactory results, as growth was impacted by bag material type as well as by suppliers of the same bag material type. However, 3D bags of 50L and above proved to be supportive of the NS0 cell line growth.

Even for cell lines that do …

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