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 …

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 …

Cell Express 100tm - A Robust, Simple And Cost Effective Alternative To Highthroughput Automated Platforms For Cell Line Development, Raj Kumar Kunaparaju

Cell Culture Engineering XV

The unrivalled growth in monoclonal antibody (mAb) therapeutics is demanding for a rapid, simple and cost effective expression system. Conventional expression system makes use of Gene Amplification Strategies, Chromatin Modifying Elements and High-Throughput Automated Platforms for cell line development. All these process are complex and costly. CELL EXPRESS 100^TM with its simple selection procedure and being able to generate 100% stable high expressing pool simplifies the isolation of high producing cell line without the need for Amplification, Chromatin Modifying Elements and High Throughput Automated Platforms. CELL EXPRESS 100^TM expression system consists of an expression vector (pUB-CE-100) and a …

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 …

Developing The Host For Targeted Integration Cell Line Development, Michelle Zhou, Isla Cheung, Shirley Yip, Mike Laird, John Joly, Brad Snedecor, Amy Shen, Yongping Crawford

Cell Culture Engineering XV

Unlike the conventional random integration (RI) cell line development (CLD), the targeted integration (TI) CLD introduces the transgene at a predetermined “hot-spot” in the CHO genome with a defined copy number (1-2 copies). Given the low copy number and the pretested integration site, TI cell lines likely exhibit better stability compared to RI cell lines. In this study, we performed a genome wide screening using transposon based cassette integration and established a TI host (255-3) that has a single landing cassette inserted in its genome. Host 255-3 was able to support the CLD for three test molecules with product titers …

Evolution Of An Integrated Continuous Antibody Manufacturing Process, Christopher Kistler

Cell Culture Engineering XV

Efforts continue to reduce the timeline and cost of progressing biologics from discovery through preclinical development to product launch. Integrated processes, those that link upstream and downstream processing with control of product quality are an attractive way to achieve meaningful reductions in the overall cost of ownership of a biologics manufacturing process.

To start along this path, an integrated continuous processing laboratory, the PROLab (Protein Refinery Operations Lab) was built to pilot the concepts of an integrated continuous antibody manufacturing process under automated control. A perfusion process was developed capable of operating for >30 days using either TFF (Spectrum KrosFlo® …

Purification Of A Hepatitis C Vaccine Candidate: Comparison Between Multi- Column Chromatographic Processes Operated In Positive And Negative Mode, Ricardo Silva, Alex Xenopoulos, Paula Alves, Manuel Carrondo, Cristina Peixoto

Cell Culture Engineering XV

Given the increasing efficiencies in bioreaction and growing interest on complex biopharmaceutical products such as virus-like particles (VLPs), downstream processing (DSP) is becoming ever more relevant. Therefore, the biopharmaceutical industry is looking for alternative downstream strategies capable of improving purification yields whilst improving product quality and lowering costs. One of most promising improvements to DSP is to replace single-column batch operation by continuous, or semi-continuous, multi-column chromatography.

We report on the development and comparison of two types of multi-column chromatographic systems aimed at the purification of Hepatitis C VLPs, produced using insect cell-based expression with recombinant baculovirus. The first process …