Engineering Commons^™

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 …

Identifying Low-Level Sequence Variants Via Next Generation Sequencing To Aid Stable Cho Cell Line Screening, Sheng Zhang, Dean Regier, David Oullette, Ivan Correia, Jerry Carson

Cell Culture Engineering XV

Developing stable Chinese hamster ovary (CHO) cell lines for biotherapeutics is an irreversible process and therefore, key quality attributes, such as sequence variants, must be closely monitored during cell line development (CLD) to avoid delay in the developmental timeline, and more importantly, to assure product safety and efficacy. Sequence variants, defined as unintended amino acid substitution in recombinant protein primary structure, result from alteration at either the DNA or the protein level. Here we report the application of transcriptome sequencing (RNAseq) in an IgG1 monoclonal antibody (mAb) CLD campaign to detect, identify and eliminate cell lines containing low-level point mutations …

Omics Approach For Generating A High-Yield Cho Cell Line Producing Monoclonal Antibodies, Wei Chi, Hsuan-Pu Chen, Dalton Chen, Hsin-Lin Lu, Bor-Shiun Chen, Chao Yi Teng, Chien-I Lin, Hsueh-Lin Lu, Chi-Chen Hsu, Sheng Jie Huang

Cell Culture Engineering XV

Chinese hamster ovary (CHO) cells are extensively used for the industrial manufacture of therapeutic antibodies. Generating high producing cell lines for secretory protein production requires knowing the bottleneck in the cellular machinery for protein expression. Integration site of gene of interest (GOI) is one of the important factors that influence the protein productivity. Even though screening of cells randomly integrated GOI can select high producing cells, the selected cell might not stable due to the chromosome instability. Here, we would like to look for host integration sites where GOI is high yield and stable by screening a single copy integration …

Crispr-Cas9 Knockout Library For Cho, Lasse Ebdrup, Alex Thomas, Hooman Hefzi, Philipp Spahn, Kaen Karottki, Helene Kildegaard, Nathan Lewis

Cell Culture Engineering XV

Traditionally, screening of large CHO cell population have been utilized to identify clones with desired phenotypic properties such as product quality, e.g. specific glyco forms, and population characteristics, e.g. ability to grow in high cell densities.

This has largely depended on the genomic variety naturally present in a large cell population or occasionally utilizing random mutagenesis to increase this variety.

The ability to precisely create genomic variety in mammalian cells have improved dramatically over the past decade and in the past few years the price has dropped substantially due to the CRISPR/Cas9 technology. E.g. knocking out a gene using CRISPR/Cas9 …

Expression Of Glycoproteins With Excellent Pharmacokinetic Properties On The Novel Cap-Go Expression Platform, Silke Wissing

Cell Culture Engineering XV

Due to their clinical importance, the development of therapeutic proteins has accelerated immensely over the past years. However, the expression of highly glycosylated recombinant therapeutic proteins like for example blood coagulation factors or serum proteins, has remained a challenging task.

C1-Inhibitor (C1-Inh) holds 7 N- and 8 O-glycosylation sites. Plasma derived C1-Inh (Berinert) as well as recombinant C1-Inh from transgenic rabbits (Ruconest) are approved for the treatment of acute attacks in patients with hereditary angioedema. However, the recombinant product shows a dramatically reduced serum half-life in pharmacokinetic studies in comparison to the plasma derived counterparts.

We have developed the …

Genetic Engineering Of Cho Cells For Viral Resistance To Mmv: Targeting Virus Binding, Internalization, Intracellular Trafficking And Transport To Nucleus, Joaquina Mascarenhas

Cell Culture Engineering XV

Contamination by the parvovirus Mouse Minute Virus (MMV) remains a challenge in CHO biopharmaceutical production processes. As part of developing a risk mitigation strategy against such events our group has evaluated the genetic engineering of Chinese Hamster Ovary (CHO) cell lines to create a new host cell line that would be resistant to MMV. While the functional receptor for MMV binding to CHO cell surface is unknown, previous work in our group has validated the role of sialic acid on the cell surface as important for cell surface binding and internalization of the MMV virus. In this work we further …

Bispecific Antibodies: Strategies, Considerations And Challenges, Christoph Spiess

Cell Culture Engineering XV

Bispecific antibodies (BsAbs) are moving mainstream as therapeutics with currently two bispecific antibodies approved and about 30 in clinical development. The idea of using a BsAb as a therapeutic has been around for almost as long as its monospecific counterpart, however during this time over 40 MAbs were approved but only two BsAbs. One of the main reasons why BsAbs lag behind is that they are far more complex to produce.

The co-expression of the two different heavy chains and two different light chains results in up to nine unwanted species in addition to the BsAb. Although a BsAb molecule …

Reduction Of Metabolic Waste Products, Ammonia And Lactate, Through The Coupling Of Gs Selection And Ldh-A Down-Regulation In Cho Cells, Soo Min Noh, Jin Hyoung Park, Gyun Min Lee

Cell Culture Engineering XV

The cultivation of Chinese hamster ovary (CHO) cells for the production of therapeutic proteins inevitably accompanies the production of metabolic wastes, mostly ammonia and lactate. Ammonia alters cell growth, productivity and the glycosylation patterns of proteins, and lactate acidifies culture media, having negative effects on cell culture. A stable CHO cell line should be established for the manufacturing process of therapeutic proteins, and the development of stable cell lines is usually based on two expression systems: the dihydrofolate reductase (DHFR) system and the glutamine synthetase (GS) system. Compared to the DHFR system, the GS system produces a reduced level of …

Development Of Hyper Osmotic Resistant Cho Host Cells, Yauharu Kamachi

Cell Culture Engineering XV

We have developed a cell culture platform for monoclonal antibody (IgG) production by Chinese Hamster Ovary (CHO) cells. The platform feed used the continuous feeding method. This platform can maintain high cell density and produce high antibody titer. However because operation of continuous feed is complex, contract manufacturing organization (CMO) that can perform continuous feed is limited. Therefore, we tried to change the feeding method from continuous feed to bolus feed. However the previous studies showed that the rapid changes of osmolality by bolus feed and the hyper osmolality repressed the cell culture growth and the final titer.

In this …

Engineering, Expression Screening, And Production Cell Line Development Of Hetero Ig Molecules Using Charge Pair Mutations, Jennitte Stevens, Guna Kannan, Trent Munro

Cell Culture Engineering XV

In recent years, there has been an increase in therapeutic indications that require bispecific targeting. Bispecific Hetero Ig antibodies that can target two antigens have long been considered as an attractive approach to drive synergistic biologic activity while maintaining the structure and stability of a traditional antibody. However, clinical development of such molecules has been hampered by CMC related challenges relating to product heterogeneity. During the development of a Hetero Ig molecule targeting the Wnt pathway antagonists Dkk-1 and SCL-1, we employed a novel strategy to drive the heterodimerization of IgG antibodies through the addition of charge pair reside mutations …

Predictive Engineering Of Cho Cells Using Systems Biology Models, Nathan Lewis

Cell Culture Engineering XV

Decades of bioprocess optimization have resulted in substantial improvements in recombinant protein production. However, some proteins remain difficult to express, and there is an increasing awareness of the need for improved control of critical quality attributes of recombinant protein drugs. To enable cell engineering efforts to enhance protein production and control product quality, we have enumerated the CHO cell parts through genome sequencing efforts,^1,2 and are now providing context to these parts by reconstructing genome-scale networks of the secretory pathway, glycosylation, and metabolism CHO (Figure 1). Using these models, which account for the activities of more than 2000 genes, …

Process Optimization For Semi-Continuous Virus Production At High Cell Densities, Daniel Vazquez, Michael Pieler, Yvonne Genzel, Ingo Jordan, Volker Sandig, Udo Reichl

Cell Culture Engineering XV

Background. Unlike production of recombinant proteins, continuous production of viral vaccines at high cell densities (HCD) is still constrained by host cell lysis during virus propagation and limited virus recovery from culture broth. Nevertheless, advanced fed-batch [1] and perfusion strategies can be applied to achieve a high-yield virus production processes. In this study, the development of a high-yield semi-continuous process for the production and purification of the modified vaccinia Ankara virus isolate MVA-CR19 and influenza A/PR/8 in HCD cultivations of the suspension cell line AGE1.CR.pIX (ProBioGen AG, Berlin) is presented.

Methods. Depending on the required scale, high cell …

Karyotype-Based Analysis Of Cell Line Instability And Clonality In Cho Cells, Jong Youn Baik, Kelvin Lee

Cell Culture Engineering XV

Chinese hamster ovary (CHO) cell line instability that can result in unexpected changes in phenotypes such as cell growth, productivity, or product quality is challenging for the biomanufacturing of therapeutic proteins. In addition, understanding cell line instability and its relationship to clonality is critical. We hypothesize that chromosomal rearrangements resulting from genomic instability are associated with cell line instability.

We developed cell line instability models using two cell lines: secreted alkaline phosphatase (SEAP)-expressing CHO cells (CHO-SEAP) and their host cells (CHO-DUK). We also developed a karyotype-based framework to quantify chromosomal rearrangements. In the absence of methotrexate (MTX), long-term cultured CHO-SEAP …

Prediction Of Stable And Transient Expression Of Recombinant Proteins From Cho Cells Based Upon Translational Reprogramming, Charlotte Godfrey

Cell Culture Engineering XV

Translational reprogramming and mRNA translation efficiency influence global protein synthesis, cell proliferation and growth; important parameters in defining recombinant protein expression yields. Reprogramming generally results in a down-regulation of overall global protein synthesis. Polysome profiling is used to analyse mRNA translation via the distribution of ribosomes between monosomes and polysomes, and can be used to investigate translational reprogramming occurring within the cell culture. Here this approach has been applied to investigate the endogenous polysome profiles of host and recombinant Chinese Hamster Ovary (CHO) monoclonal antibody-producing cell lines, and how the profiles change across culture depending on the growth and protein …

Establishing A Robust Two-Step Cloning Strategy For The Generation Of Cell Lines With A High Probability Of Monoclonality, Alison Young, Claire Lovelady, Mahesh Shivare, Jarka Glassey, Shirley Coleman, Alison Porter

Cell Culture Engineering XV

A regulatory requirement for the production of therapeutic proteins from mammalian cells is that the production cell line is clonal, that is, derived from a single progenitor cell. It is therefore standard procedure to include at least one cloning step during the development of a recombinant cell line for therapeutic protein production. Numerous techniques can be employed for cloning cell lines, but regardless of the cloning method used there should be appropriate evidence to support that the method is fit for purpose. A point highlighted by the increasing interest from regulatory bodies regarding the cloning method used and the probability …

Identifying Opportunities In Cell Engineering For The Production Of ‘Difficult To Express’ Recombinant Proteins, Hirra Hussain, Alan Dickson, Mark Abbott, Robert Roth, David Fisher

Cell Culture Engineering XV

There is a growing demand for production of recombinant proteins of many structural varieties in mammalian expression systems, either as therapeutics or for protein characterisation. However, certain recombinant proteins are “difficult to express” in mammalian expression systems requiring extensive cell line and process optimisation which, as a result, can have significant consequences for drug development processes. The Tissue Inhibitors of Metalloproteinase (TIMP) protein family, TIMP-2, -3 and -4, are naturally secreted proteins that share significant structural homology (~50% identity and ~70% similarity in amino acid sequence), but show profound differences in secretion in mammalian expression systems. Computational sequence analysis of …

Accelerated Homology-Directed Targeted Integration Of Transgenes In Cho Cells Via Crispr/Cas9 And Fluorescent Enrichment, Jae Seong Lee, Lise Marie Grav, Gyun Min Lee, Helene Kildegaard

Cell Culture Engineering XV

Development of recombinant CHO cell lines has been hampered by unstable and variable transgene expression caused by random integration. With draft genome of several CHO cell lines and targeted genome editing technologies, rCHO cell line development based on site-specific integration has the potential to overcome the limitations of clonal heterogeneity. In a previous study, we demonstrated efficient and precise targeted gene integration in CHO cells using CRISPR/Cas9 technology and homology-directed repair (HDR) pathway¹⁾. However, it requires a selection process, which limits targeted integration of multiple transgenes at multiple sites due to a limited number of selection markers and …