Engineering Commons^™

Rapid Development Of A Perfusion Process With High Productivity, Sen Xu, Linda Hoshan, Balrina Gupta, Hao Chen

Cell Culture Engineering XV

Continuous process improvement is required during a biologics process life cycle to increase process yield and reduce cost. In this poster, we will present a case study in late stage process development where everything was changed but the product quality. The following major changes were made when developing the new process:

• Production cell line – subclone of the original clone used.
• Media – from hydrolysate-containing feeds to fully chemically-defined feeds.
• Feeding strategy – from fixed volume/fixed schedule feeding to daily on-demand feeding.

Media components were optimized through spent media analysis and mechanistic understanding to accommodate the new feeding strategy. High-throughput …

Bioanalytical Comparability Of Biotechnology Products Subject To Changes In Their Manufacturing Process, Jose Menezes, Maria Campos, Pedro Felizardo

Cell Culture Engineering XV

Over lifecycle, from development through all industrialization stages and then over manufacturing history across multiple sites, a biotechnology product must show very high levels of quality consistency, as that is a pre-requisite for safety and efficacy to patients (cf. ICH Q5E, 2004).

Bioanalytical similarity has acquired over the last years another meaning with the introduction of biosimilars: products that in principle cannot be identical to their reference products as processes used and other design options are not exactly identical, leading for example to minor differences in clinically inactive components. However, biosimilars must be proven to be pharmacologically identical to their …

Real Time Prediction And Control Of Glycoform Profile Of Mammalian Cell Cultures Using In Silico Glycosylation Model Coupled With Extracellular Metabolites, Sha Sha, Seongkyu Yoon

Cell Culture Engineering XV

In silico mechanistic models for glycosylation are investigated for biotherapeutics development and production. The models systematically describe the transport-kinetics of glycosylation process and serve as a prediction framework for glycan profile. However, inputs for those models are generally limited to intracellular parameters, such as enzymatic activity and nucleotide sugar concentration. These intracellular metabolites are unfortunately inaccessible in process. To modulate those models for glycoform prediction in culture process, linkage between measurable culture variations and the intracellular metabolites change will be thoroughly investigated. The measurable metabolites and intracellular variables associated with in silico glycosylation model are integrated in the modeling. Despite …

Scalability Of The Mobius® Single-Use Bioreactor From Bench To Clinical Scale: Examination Of Key Engineering Parameters And Robustness, Lee Madrid

Cell Culture Engineering XV

To successfully implement a large scale single-use bioreactor platform, equivalent performance, i.e. scalability, with smaller sized bioreactors across the platform must be demonstrated. With the recent development of a single-use clinical scale (2000 L) bioreactor, the EMD Millipore bioreactor portfolio now spans from the bench-scale (3 L), through the small-scale (50 L) and pilot-scale (200 L) up to the manufacturing process volume range. The performance design space of the entire Mobius® Single-use Bioreactor platform was characterized using several key engineering parameters including oxygen mass transfer coefficient (kLa), power per unit volume, Reynolds number (Re), mixing time and tip speed. Based …

Overcoming Scale-Up Challenges With A Non-Robust Cell Line, Sigma Mostafa, Brian Baker, Abhinav Shukla

Cell Culture Engineering XV

Cell culture scale up success depends on robust cell line, process, and equipment. Mixing characteristics of bench scale through large scale bioreactors need to be well understood and a reliable scale up parameter needs to be used. The cell culture raw material lot to lot variability as well as hydration protocol reliability needs to be considered. The cell culture process parameter ranges need to be broad enough to account for system variability and the process should not run at the edge of failure. In our experience, greater than 95% of cell lines are robust and easily scalable. Cell line robustness …

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

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 …