Biomedical Engineering and Bioengineering Commons^™

Development Of Bioreactor Auto-Sampling System For Real Time Product Quality Monitoring In Mammalian Cell Culture, Meena George

Cell Culture Engineering XV

Product quality attributes such as glycosylation, size and charge variants are critical characteristics for the development of biotherapeutics in mammalian cell culture processes in order to target a desired product profile. In an attempt to characterize the entire time-course of a fed-batch culture under different media conditions, a bioreactor autosampling device integrated with cell removal and sample retention systems and at-line instruments such as Patrol UPLC was developed to rapidly analyze the cell culture IPC as well as product quality changes in real time. This information can be rapidly translated for better process understanding and process control to enable optimization …

Accelerate Cell Culture Development Using The Modular Automated Sampling Technology (Masttm) Platform In An Integrated Bioprocess Lab Environment, Clinton Pepper

Cell Culture Engineering XV

Biopharmaceutical companies are continually striving to fundamentally understand and optimize cellular performance within their bioreactors. While on-line process analytical technology (PAT) tools like dielectric and Raman spectroscopy are helping to provide insight into cell processes that impact titer and antibody quality, the integration of these data with off-line measurements such as cell density, viability, titer and glycosylation remains an elusive goal.

Bend Research Inc., in collaboration with Lilly, Pfizer and other major biopharmaceutical companies, is advancing the Modular Automated Sampling Technology (MAST^TM) platform and integrating this system with automated analytical and data retrieval systems. This approach allows application …

Kinetic Physico-Chemical Model For Cell Culture Processes – Applications And Opportunities, Natraj Ram, Kartik Subramanian, John Ponzo

Cell Culture Engineering XV

Cell culture processes for production of recombinant proteins can be modeled to provide critical insights into the interrelationship between several parameters that impact the process performance and control. For the first time, we describe a model that incorporates pH control in combination with gas transfer to provide a more complete description of the physicochemical processes that occur during the entire course of the cell culture process. The model includes multi-component chemical equilibria involving carbonate, lactate and sodium hydroxide coupled with electroneutrality for calculation of pH. Further, the pH feedback control has been incorporated depending on levels of carbonate and lactate …

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 …

Poly-Pathway Model Approach: Simulation Of Multiple Metabolic States, Erika Hagrot

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 …

Improving The Productivity And Product Quality Of Antibodies Expressed From A Cho Transient System, Athena Wong

Cell Culture Engineering XV

Transient transfection of CHO cells is a widely used tool to express recombinant proteins for non-GLP preclinical studies due to the system’s ability to rapidly produce proteins of similar product quality to CHO stable cell lines. Our high throughput CHO transient transfection process allows for efficient generation of milligram to multi-gram amounts of protein from small scale automated 96-deep well plates and tubespins to large scale 100L bioreactors. In this work, we present a case study describing the influence of media components on PEI-mediated CHO transfections. Given the dependence of PEI transfection on electrostatic interactions for formation and cell internalization …

Cho-Specific Recombinant Protein Glycosylation Reaction Network, Benjamin Kremkow, Kelvin Lee

Cell Culture Engineering XV

Protein glycosylation is one of the most important product quality attributes and impacts efficacy, half-life, and immunogenicity. Previous glycosylation models effectively simulated the key parts of the N-glycosylation pathway. Building upon these prior efforts as well as recent CHO-K1 and Chinese hamster (CH) genome sequencing efforts, we share a new model for CHO- and CH- glycosylation. The model contains all N-glycosylation-related genes and all of the metabolic genes associated with central carbon metabolism, nucleotide sugar precursor synthesis, and nucleotide sugar transport annotated from the CHO-K1 and CH genomes. The model predicts both intracellular and secreted glycans for both mAb and …

Lensless Imaging For Continuous Cho Viable Cell Density Monitoring In Bioreactors, Geoffrey Esteban, Martin Pisaneschi, Jeremie Cubeta, David Sergeant

Cell Culture Engineering XV

During suspension cell cultures in bioreactors, traditional measure for cell count and cell viability still rely on sampling and staining protocol where the Trypan Blue exclusion method is performed once a day. While automatic cell counters have reduced the statistical error of the original manual method, sampling the bioreactor is a risk for contamination and is prohibiting the use of such method for process control as the sampled volume becomes significant. Lensless Imaging Technology is a new breakthrough method for accurately and precisely determine cell concentration and viability without staining. This technique has the unique capability of acquiring microscopy images …

Evaluation Of Product Antibody (Mab) Heterogeneity In Non-Clonal Cell Pools For Early Pre-Clinical Development, Gabi Tremml

Cell Culture Engineering XV

During early stage of pre-clinical biologics development, grams of product antibody is needed for process development, formulation development, and analytical assay development. To accelerate the pre-clinical timeline, it's a common practice to use master wells (non-clonal stable cell pools) to generate development material. The advantage of using master wells is that the product antibody is generated from the same host cell line and expression vector in an earlier and shorter time. Thus, the purified product antibody can be representative to the final therapeutic product. However, the non-clonal nature of the cell pools can give rise to potential risk of heterogeneity …

Adjusting Product Quality Attributes Of A Biosimilar Using Process Levers, Brett Belognia

Cell Culture Engineering XV

The development of a biosimilar introduces many unique challenges compared to that of a novel drug. The primary consideration in developing a biosimiliar is that the product quality of the protein of interest be “highly similar” to that of the branded product. Product quality attributes are defined by the manufacturing cell line and the manufacturing process. The ability to manipulate the manufacturing process is key to obtaining a highly similar product. This poster will focus on the development of biosimilars and provide examples of how the product quality can be manipulated with process levers to achieve similarity with that of …

Investigating The Reverse Warburg Effect: How High Extracellular Lactate Alters Breast Cancer Metabolism, Daniel Odenwelder, Sarah Harcum, Arthur Brodksy

Cell Culture Engineering XV

Metabolism plays a critical role in the progression of cancer malignancy, with nutrient source flexibility allowing cancer cells to outcompete normal cells and survive harsh environmental conditions. Lactate accumulation in poorly vascularized tumors has predominately been considered a waste product resulting from the Warburg effect; however, recent studies have shown that lactate can additionally serve as a metabolic substrate for some cancer cells, a process referred to as the Reverse Warburg effect. Lactate accumulation is also associated with cancer stem cell-like gene expression, drug-resistance, metastasis, and poor prognosis in breast cancer patients. Unfortunately, little is known about the role of …

Advanced Process Monitoring And Feedback Control To Enhance Cell Culture Process Production And Robustness, An Zhang, Valerie Tsang, Brandon Moore, Vivian Shen, Yao Ming Huang, Rashmi Kshirsagar

Cell Culture Engineering XV

It is common practice in biotherapeutic manufacturing to define a fixed-volume feed strategy for nutrient feeds based on historical cell demand. However, once the feed volumes are defined, they are inflexible to batch-to-batch variations in cell growth and physiology and can lead to inconsistent productivity and product quality. In an effort to control critical quality attributes and to apply Process Analytical Technology (PAT), we demonstrated three different and novel approaches for implementing online monitoring and feedback control to improve the performance and/or robustness of cell culture processes. First, we describe the first reported fed-batch process utilizing online amino acid measurements …

Implementation Of A Recirculating Tff N-1 Perfusion System At Manufacturing Scale: Conquering Process Hurdles And Scaling Challenges, Alex Doane, Weiwei Hu, William Yang, Kelly Wiltberger, Haofan Peng

Cell Culture Engineering XV

In order to increase the output of a traditional large scale stainless steel fed-batch manufacturing facility without major engineering work, we investigated the implementation of N-1 perfusion technology to feed a more intensified fed-batch production process. Perfusion N-1 enables cells to grow to high densities in the seed train bioreactor. By shifting cell growth from production to seed train, we can increase seed train occupancy per batch and reduce production bioreactor occupancy per batch, which improves facility throughput by bringing the run duration ratio of N-1/N closer to unity. Instead of using the popular ATF technology, an in-house TFF system …

Efforts To Reduce Impact Of Media Variability On Product Quality For A Commercial Perfusion Process, Nirel Rillera, Kevin Yeh, Benjamin Youn, Jerome Acuna, John Henstrand, Sean Forestell

Cell Culture Engineering XV

Strict control of raw materials used in bioprocesses is necessary to ensure consistent product quality. However, use of poorly characterized complex components makes achieving such strict control difficult. A strong correlation between product quality variability among bulk drug substance lots and changes in a complex medium component was observed in a commercial perfusion process. This correlation was subsequently confirmed experimentally at both large and small scales. A multifaceted strategy was applied to investigate and mitigate the impact of the media variability on product quality, which included (1) studies in small scale perfusion bioreactors; (2) development of a simple cell based …

To Clone Or Not To Clone? – Wrong Question! An Investigation On Genome Heterogeneity And Stability And On What Controls Cell Behavior, Nicole Borth

Cell Culture Engineering XV

The most striking characteristic of CHO cells is their adaptability, which enables efficient production of proteins as well as growth under a variety of culture conditions, but also results in genomic and phenotypic instability. Potential causes include i) the high number of chromosomal rearrangements, including variation in chromosome numbers observed in CHO or any other rapidly growing cell line; ii) mutations including SNPs or InDels that change the activity or function of enzymes; iii) epigenetic changes that alter the gene expression pattern of a cell without impacting the genome sequence itself. To understand the relative contribution of these towards phenotype …

A Systematic Development Approach To Optimize And Control Biopharmaceutical Product Quality, Min Zhang, Leisha Youngblood, Carrie Weymer, Gian Arias, Tim Hill

Cell Culture Engineering XV

The complexity of recombinant protein manufacturing processes, including expression systems (i.e. host cell line, expression vector, cell line engineering process), cell culture process conditions, and choice of media and feeds, present significant challenges to achieve desired product quality of biopharmaceuticals. Significantly more challenging is to develop processes for biosimilar proteins, which must match product quality attributes of the originator molecule, show comparable in-vitro biological activity, and have no clinically meaningful differences as compared to innovator products. To address these challenges, and provide appropriate, flexible, and quick solutions to govern product quality control during manufacturing, Fujifilm Diosynth Biotechnologies (FDB) has developed …

Building Quality Novel Formats And Development Processes, Steven Lang

Cell Culture Engineering XV

There has been an explosion of interest in bispecific or multi-specific bio therapeutic formats. Monospecific antibodies remain the foundation for most bispecific formats. However, monospecific strategies cannot be directly translated into the discovery and development of bispecific antibodies. Bispecifics present unique challenges especially for efficient and consistent assembly/production processes and analytical methods to ensure high quality drug products. Improvements in selection of bispecific combinations/architecture, yield and efficiency and common analytical issues and resolutions will be discussed. Applying improved methods learned from Fab-arm exchange bispecifics and alternative scaffold-antibody fusions, we have established a foundation of knowledge that enables accelerated development

Generation Of A Stable Pluripotent Cell Line From Chinese Hamster Embryonic Fibroblasts, Haiyun Pei, Dong Seong Cho

Cell Culture Engineering XV

Chinese hamster ovary (CHO) cells are aneuploidy and exhibit a high degree of genomic instability. Many studies having a cell line with a stable karyotype and genome structure is highly desirable. We have derived a stable cell line from Chinese hamster embryonic fibroblasts by transduction of mouse transcription factors M3O (a fusion gene which is chimeric of Oct4 and the effective transactivation domain of MyoD), Sox2, Klf4, and n-Myc using a lentivirus vector. The cells showed morphology of typical murine pluripotent stem cells, stained positively with alkaline phosphatase, expressed endogenous Sox2, Nanog and surface antigen SSEA1, resembling mouse embryonic stem …

Assessment Of Genomic Instability In Chinese Hamster Ovary (Cho) Cells, Sabine Vcelar, Martina Baumann, Michael Melcher, Nicole Borth

Cell Culture Engineering XV

CHO cells are the number one production system for therapeutic proteins due to their ease of handling, their fast growth in suspension culture and their capability to perform complex protein folding and human-like post-translational modifications. This flexibility is in part due to, but at the same time set off by the frequent occurrence of chromosomal rearrangements and other genomic variants, which influences individual cell line performance and the stability of industrial producer cell lines, resulting in prolonged screening phases in order to isolate cells with sufficiently stable properties. Furthermore producer cell properties are also frequently lost again over time and …

A Novel Bispecific Antibody For Her2+ Breast Cancer: The Beat Gbr 1302, Pierre Moretti, Julie Macoin, Amelie Croset, Darko Skregro, Romain Ollier, Stanislas Blein, Martin Bertschinger, Samuel Hou, Jonathan Back

Cell Culture Engineering XV

While the idea of bispecific drugs was brought up over 30 years ago, the development of formats mature enough for the clinic remained for a long time a challenge. The whole field has been hampered by major problems of manufacturability (e.g. product purity and yields) and immunogenicity. With the recent arrival of new bispecific formats, either as antibody–like molecules (containing an Fc) or scFv fragments, at least 18 bispecific molecules have entered clinical trials showing very promising results. The BEAT® format has been developed as bispecific antibodies maintaining the pharmacokinetics and the low immunogenicity of human IgG with excellent manufacturability …

Automated, High Throughput Imaging During Cell Line Development To Increase The Assurance Of Clonality, David Shaw

Cell Culture Engineering XV

Current methodologies to create monoclonal cell lines include limiting dilution or single-cell sorting at conditions that offer statistical assurance of monoclonality. We have implemented an automated, high-throughput imaging workflow that acquires brightfield and fluorescent images of every well during the single-cell cloning process. These images can provide direct evidence on whether the cell line originated from one cell during the cloning step. Characterization of the workflow was completed, including the effect of fluorescent dye on cell culture, effect of exposure to high intensity light on cell culture and rate of random mutagenesis in the presence of fluorescent dye and high …

Genome-Wide Rnai Screen For Improved Functional Expression Of Recombinant Proteins From Hek 293 Cells, Joseph Shiloach

Cell Culture Engineering XV

For the purpose of improving recombinant protein production from mammalian cells, an unbiased, high-throughput whole-genome RNA interference screen was conducted using human embryonic kidney 293 (HEK 293) cells expressing firefly luciferase. 21,585 human genes were individually silenced with three different siRNAs for each gene. 56 genes whose silencing caused the greatest improvement in the luciferase expression were found to be part of several different pathways that are associated with spliceosome formation/mRNA processing, transcription, metabolic process, transport and protein folding. 10 genes whose downregulation significantly enhanced the protein expression were validated by their silencing effect on four different recombinant proteins. Among …

Microfluidic Accelerated Evaluation Of Cho Cell Clones By Perfusion Of Fed-Batch Conditioned Media, Darek Sikorski

Cell Culture Engineering XV

The generation of genetically engineered production CHO cell lines is normally the longest step in the race to scale-up protein manufacturing. This labor-intensive screening includes the expansion of hundreds of clonal cultures to sufficient numbers for their growth and productivity to be evaluated. Unfortunately, many clones that perform well when screened at the batch cloning stage display reduced performance under fed-batch conditions. Thus, screening potential clones under conditions more similar to the ultimate production cultures provides an opportunity for more effective clone selection.

We combined the advantages of precise measurements in microfluidic nL volume cultures with our ability to modify …

Cell-Controlled High-Intensity Perfusion And Hybrid Fed-Batch Systems That Drastically Reduce Perfusion Rates And Harmonize With Continuous Downstream Processing, Gregory Hiller, Matthew Gagnon, Ana Maria Ovalle, Bhanu Chandra, Wenge Wang, Elizabeth Eydelman

Cell Culture Engineering XV

Peak cell densities in fed-batch cultures of mammalian cells can be limited by the depletion of nutrients or accumulation of inhibitors. Since addition of nutrients is relatively straight forward, inevitably it is growth inhibitor buildup, combined with the practical limits of feed volume addition and the problem of amino acid counter ion and miscellaneous osmolyte accumulation, that serve to limit productivity in the industry standard fed-batch bioreactor. To break these barriers we have begun to investigate mixed modes of bioreactor operation, some using novel methods for cell-controlled perfusion, which can substantially reduce the volumes of perfusion medium required, suppress lactic …

Flow Cytometry Screening Strategy For The Enrichment Of High-Producing Chinese Hamster Ovary Cells For Monoclonal Antibody Manufacturing, Takeshi Okumura

Cell Culture Engineering XV

Establishment of Chinese hamster ovary (CHO) cell lines for the production of monoclonal antibodies (mAbs) is generally conducted through transfection and pooling procedures with a laborious and time-consuming screening. It could be one of the most rate-limiting steps in the process development of mAb manufacturing. Therefore, an improvement to screening efficiency will give a positive impact to shortening the period of process development. We here propose an enrichment strategy for screening for high-producing cells using flow cytometry (FCM).

A stable pool prepared by mAb-expression vector transfection was stained with a fluorescent-labeled antibody that binds to an mAb presented on the …

Systems Engineering Of A Cho Cell Line For Enhanced Process Robustness, Kyoungho Lee, Hsu-Yuan Fu, Liang Zhao, Lei Feng, Tung Le, Haiyun Pei, Sugandha Rajput, Dong Cho, Nikunj Somia, Scott Mcivor, Wei Hu

Cell Culture Engineering XV

Recent advances in genome engineering have opened great opportunities for engineering Chinese hamster ovary cells. An ideal cell line is no longer just one with high productivity, but also with high stability in both the productivity and product quality, and in both extensive passaging and long term continuous culture. Furthermore, an ideal cell line should be provided with process controllability, allowing the use of environmental control variables to steer its metabolism to a reaction pathway that favors the synthesis of product with the desired quality attributes. Importantly, these superior traits must be genetically and epigenetically stable and be passed on …

Time Course Of Transcription And Chromatin States During Batch Culture In Chinese Hamster Ovary Cells, Inmaculada Hernandez

Cell Culture Engineering XV

Chinese Hamster Ovary (CHO) cells are known to be easily adapted to different culture conditions and to be highly variable. Beside genetic alterations, epigenetic regulation, such as histone modifications, can play a role in determining altered cell and production characteristics. Epigenetic control is mainly achieved by DNA-methylation and modifications of histones. The main effect of these modifications is a change in chromatin structure, influencing the transcriptional machinery at the respective locus. So far, there is very little information available about changes in epigenetic regulation in cells in response to altered substrate availability and culture environment.

Here we examine the gene …

Effective Micrornas For Cell Line Engineering And Cellular Mechanisms Of Action, Kerstin Otte, Albert Paul, Rene Handrick, Simon Fischer, Verena Emmerling

Cell Culture Engineering XV

MicroRNAs (miRNAs) constitute an important class of small non-coding RNAs and are imperative for the regulation of gene expression in mammalian cells. By post-transcriptionally modulating the expression of hundreds of different genes concomitantly, miRNAs are capable of regulating entire cellular pathways to control cellular phenotypes. Thus, miRNAs represent promising tools for cell engineering of biopharmaceutical production cells.

By taking advantage of a functional high-content miRNA screening procedure we were able to identify >750 miRNAs significantly affecting protein expression, cell growth, apoptosis and necrosis in CHO cells. Functionality of impactful miRNAs could be successfully validated in secondary screening approaches as well …

Re-Programming Cho Cell Metabolism Using Mir-23 Tips The Balance Towards A Highly Productive Phenotype, Niali Barron

Cell Culture Engineering XV

microRNA-mediated engineering of CHO cells is an attractive method for enhancing various industrially relevant phenotypes, ultimately boosting recombinant protein titres. A single miRNAs ability to interact with multiple mRNA targets allows their regulatory capacity to extend to processes such as cellular metabolism. Various metabolic states have previously been associated with particular CHO cell phenotypes such as glycolytic or oxidative metabolism accommodating growth and productivity, respectively. miR-23 has previously been demonstrated to play a role in glutamate metabolism resulting in enhanced oxidative phosphorylation through the TCA cycle. Re-programming cellular bioenergetics through miR-23 could tip the balance, forcing mammalian production cells to …