Biomedical Engineering and Bioengineering Commons^™

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 …

Monitoring Live Stem Cells In Suspension And Attached To Carriers In Conventional And Single Use Bioreactors, John Carvell, Aditya Bhat

Cell Culture Engineering XV

Embryonic stem cells are promising cell sources for regenerative medicine and organ replacement after disease or injury. Traditional single use bioreactors such as the Sartorius Biostat Rocking Motion bag are being used for growing up these cells at the larger scale and a number of companies are developing fully automated, closed bioreactor systems specifically for growth of stem cells for clinical applications eg Xpand (Holland), Pall(USA).

The stem cells in the bioreactors need to be monitored and controlled for both product quality and to satisfyGMPrequirements and one of the most important parameters is the concentration of live cells. On-line monitoring …

Influence Of Cultivation Parameters Or Supplement On Product Qualities And Culture Performances During Perfusion, Kyu Kim, Sam Seol, Soo Ryu, Sang Park, Sun Kim, Seung Lee

Cell Culture Engineering XV

Perfusion processes have been developed with technological advances in single-use bioreactor and cell retention device. Perfusion has advantages such as high cell density culture in compact facilities and media change or culture parameter shifts for protein production with desired qualities. Single use bioreactor (200L or 1000L) with ATF System was used for the clinical production. For the process development, the scale-down model was established with lab-scale (2L) bioreactor with ATF2.

A recombinant CHO cell line producing a fusion protein was cultivated using in-house serum-free media. Influence of insulin (0~3mg/L) on qualities was investigated in the established perfusion process using in-house …

Elucidating Glycosylation Pattern Of Protein Produced In Mammalian Cells, Tung Sy Le

Cell Culture Engineering XV

The glycosylation pathway is a highly branched network. Although only a relatively small number of enzymes are involved in the pathway, a multitude of intermediate and terminal glycans can be formed. Each intermediate glycan in the network can be the substrate of subsequent glycosylation steps. In each step, there can be multiple enzymes acting on a single glycan due to overlapping substrate specificity. These enzymes tend to compete with each other to divert the network flux toward a particular branch. Alternation of their relative ratios can result in different glycan profiles. In mammals, such enzymes are distributed in a tissue-specific …

Reduction Of N-Glycan Profile Variation By Using Capacitance Probes For Optimized Process Control, Christopher Bro, Tom Kristensen, Michael Wahlers, An Zhang, Anne Tollstrup

Cell Culture Engineering XV

The glycan profile of therapeutic monoclonal antibodies frequently plays an important role in their biological function and pharmacokinetics. Therefore, improved control of the glycosylation profile of biopharmaceutical monoclonal antibodies has become an increased priority during late stage and commercial manufacturing of New Biological Entities as well as biosimilars. Two ways to obtain better control are through process parameter optimization and/or through addition of media supplements to the production reactor. Cell culture supplementation with mycophenolic acid is one method to efficiently manipulate N-glycan profiles of monoclonal antibodies, notably the level of fucosylation. We have observed at least for some CHO-based cell …

Impact Of Culture Conditions And Cell Age On Sequence Variant Levels In Monoclonal Antibody Biotherapeutics, Karin Anderson

Cell Culture Engineering XV

Sequence variants (SV) are protein products that contain unintended substitutions in the amino acid sequence. SV are one source of heterogeneity inherent to biological systems. They can arise from multiple sources within CHO cells including mutations in genomic DNA, transcription and/or translation errors, splice variants, and post translational modifications. This poster focuses on SV resulting from single base changes in a copy of the integrated exogenous gene in genomic DNA of single cell cloned CHO cell lines.

The increased sensitivity and selectivity of analytical instruments and methods has revealed across industry that a number of cloned CHO cell lines, including …