Biomedical Engineering and Bioengineering Commons^™

Integrated Strategies For The Production, Maturation And Storage Of Functional Cardiomyocytes Derived From Human Pluripotent Stem Cells, Claudia Correia, Paula Alves

Cell Culture Engineering XV

The production of cardiomyocytes (CM) from human pluripotent stem cells (hPSC) holds great promise for cardiotoxicity drug testing, disease modeling and cardiac regeneration [1]. However, the complex nets of signalling pathways involved in cardiomyogenesis compromises the effectiveness of the existing differentiation protocols to reproducibly produce high-quality CM from hPSC (hPSC-CM). Produced hPSC-CM are immature compared with adult CMs, express typical fetal cardiac genes, have immature electrophysiological properties and use glucose as major energy source [2]. The applicability of hPSC-CM in the clinic/industry is also dependent on the development of efficient methods for worldwide shipment of these cells. In this study …

Modelling Short-Latency Disparity-Vergence Eye Movements Under Dichoptic Unbalanced Stimulation, Agostino Gibaldi, Guido Maiello, Peter J. Bex, Silvio P. Sabatini

MODVIS Workshop

Vergence eye movements align the optical axes of our two eyes onto an object of interest, thus facilitating the binocular summation of the images projected onto the left and the right retinae into a single percept. Both the computational substrate and the functional behaviour of binocular vergence eye movements have been the topic of in depth investigation. Here, we attempt to bring together what is known about computation and function of vergence mechanism. To this aim, we evaluated of a biologically inspired model of horizontal and vertical vergence control, based on a network of V1 simple and complex cells. The …

Measuring And Modeling Shared Visual Attention, Jeffrey B. Mulligan, Patrick Gontar

MODVIS Workshop

Multi-person teams are sometimes responsible for critical tasks, such as flying an airliner. Here we present a method using gaze tracking data to assess shared visual attention, a term we use to describe the situation where team members are attending to a common set of elements in the environment. Gaze data are quantized with respect to a set of N areas of interest (AOIs); these are then used to construct a time series of N dimensional vectors, with each vector component representing one of the AOIs, all set to 0 except for the component corresponding to the currently fixated …

Challenges In The Use Of Scale-Down Models For Understanding And Mitigating Process Variations Of A Monoclonal Antibody Production Process, Peter Russo

Cell Culture Engineering XV

Scale-down models are commonly used to execute process characterization studies, as well as to screen raw materials and to conduct investigations in support of manufacturing operations. During the clinical manufacture of a monoclonal antibody, consistent product quality was maintained; however, large variations in bioreactor harvest titer (> 2X) were observed between lots. Root cause analysis of this variation did not establish a linkage between culture performance and manufacturing execution or deviations. Further, small scale satellite reactor performance displayed lesser variability; thereby eliminating seed train and raw materials as potential root causes. Small scale investigations, using multiple scale-down models, were not …

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 …