Biomedical Engineering and Bioengineering Commons^™

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® …

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 …

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 …

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 …

An Integrated Cell Line Development Platform For Generation Of High Yielding Cho Stable Cell Lines Expressing A Stabilized Trimeric Pre-Fusion Rsv F Recombinant Viral Glycoprotein, Richard Schwartz, Amritha Menon, Mingzhong Chen, Giulia Fabozzi, Althaf Hussain

Cell Culture Engineering XV

Accelerating timelines to deliver stable cell lines with high productivity is challenging, especially for biologics portfolios which include complex recombinant vaccine constructs. The VRC’s current integrated approach to shorten CHO cell line development timeline utilizes host cells pre-adapted to production medium, optimization of the expression vectors, improved pre and post-transfection methodology, automated ClonePix^TM technology and top clone selection and ranking in micro-scale ambr bioreactor technology. This platform was developed with monoclonal antibodies as model proteins. While this integrated platform enabled generation of high yielding clones expressing monoclonal antibodies, it was initially much less successful with a difficult to express …

Generation Of Desirable Cho Cell Factories With Predictive Culture Performance Using Crispr/Cas9-Mediated Genome Engineering, Helene Faustrup

Cell Culture Engineering XV

Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry as a host for the production of complex pharmaceutical proteins. Thus, genome engineering of CHO cells for improved product quality and yield is of great interest. Here, I will demonstrate our latest advances in improving the efficiency of CRISPR/Cas9-mediated genome engineering to generate attractive knockout and knockin CHO cell lines. Analysis of the dynamics and efficiency of the technology will be demonstrated for genes involved in glycosylation and apoptosis. Combined with multiplexing and fluorescent enrichment, application of CRISPR/Cas9 genome editing facilitated disruption of several genes simultaneously and accelerated …

A Novel Platform For High Throughput Cell Line Screening & Development, Maria Wendt, Christoph Freiberg, Cristopher Smith, Hans Fischer

Cell Culture Engineering XV

The provision of stably expressing, high-yield mammalian cell lines is a key step in bioprocess development. Recent progress in clone screening automation, and the increased use of mini-bioreactors, such as ambr^®, have resulted in higher experimental throughput and a significant increase in data that needs to be handled and interpreted.

Co-developed in close collaboration with leading biopharmaceutical companies, we have implemented a dedicated cell line development platform for fully automating the clone line selection and assessment process to increase process efficiency and quality. The new platform supports the entire cell line development workflow including seeding, selection, incubation, passaging, …

Sub-Physiological Culture Temperature Boosts Expression Levels Of Membrane Proteins In Cho Cells, Sampath Kumar, Lihui Zhong, Beata Bobrowicz, Juergen Nett

Cell Culture Engineering XV

Membrane proteins especially G-protein coupled receptors (GPCR) are the targets of a large number of small molecule therapeutics. With the advances in biopharmaceutical therapeutic approach, development of antibodies against the membrane proteins are increasingly attempted. Generation of therapeutic antibodies against these targets is a major challenge due to the limited accessibility of these proteins in vivo. Stable clonal cell lines expressing the membrane proteins are used as antigen-presenting cells for antibody development in one of two ways: whole-cell based selection including complexing with B cells, or immunization. They also serve as valuable reagents in characterizing the binding properties of …

Challenges Developing Biologics For The Prevention And Treatment Of Infectious Diseases Impacting Global Health, Steven Hadley

Cell Culture Engineering XV

Biologics have enjoyed tremendous success as products in high income countries of the world. However the high cost of these products today prevent their use in low income countries and with the notable exception of palivizumab there has been little commercial interest in developing biologics for infectious diseases that are prevalent in these geographies. Recent advances in the study of HIV, malaria, pertussis and Ebola highlight the potential of monoclonal antibodies (mAb) for prevention or treatment of these diseases. The evolution of the industrialized CHO process over the past 30 years to satisfy market demands for mAbs is remarkable. The …

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 …

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 …

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 …

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 …

Proof That Can Travel - Documented Clonality Report For Regulatory Submission, Ian Taylor, Paul Miller

Cell Culture Engineering XV

Clonality is a key element of cell line development and is an important component of a regulatory submission. Indeed for BLA, the clonality of MCB is mandatory. Historically, the regulator has insisted upon 2 rounds of cloning for developing a cell line and assurance of monoclonality based on statistical outgrowth measurements. Recent improvements in high resolution whole well imaging of cells in microplates, enables the creation of indisputable image-based evidence for the growth of a colony from a single cell. This evidence eliminates a round of sub-cloning and results in several months of cell line development time being saved. However, …

Overcoming Process Intensification Challenges To Deliver A Manufacturable And Competitive Integrated Continuous Biomanufacturing Platform, Jason Walther, Neha Shah, Myles Hollenbach, Jonathan Wang, Marcella Yu, Jiuyi Lu, Yang Yang, Konstantin Konstantinov, Chris Hwang

Cell Culture Engineering XV

Groups in both industry and academia have achieved high densities and productivities in perfusion cell culture processes. At Sanofi, we have demonstrated perfusion densities greater than 100 million cells/mL (with associated high productivities) at a cell-specific perfusion rate of only 20 pL/cell/day. This process intensification reduces the footprint of upstream unit operations as well as capital and operating expenses of manufacturing facilities. The continuous nature of perfusion cell culture also creates opportunities for integration of continuous downstream operations, leading to further process intensifications and volume reductions.

In this presentation, we will discuss our work on several upstream challenges that must …

Cre-Loxp-Controlled Cell-Cycle Checkpoint Engineering In Chinese Hamster Ovary Cells, Takeshi Omasa, Rima Matsuyama, Tomomi Tsusui, Noriko Tamano, Masayoshi Onitsuka, Kyoung Lee

Cell Culture Engineering XV

The gene amplification system is widely used in Chinese hamster ovary (CHO) cells for the productive cell line construction of therapeutic proteins. To enhance the efficiency of conventional gene amplification systems, we previously presented a novel method using cell-cycle checkpoint engineering¹⁾. Here, we constructed high-producing and stable cells by the conditional expression of mutant cell division cycle 25 homolog B (CDC25B) using the Cre-loxP system²⁾. A bispecific antibody-producing CHO DG44-derived cell line was transfected with floxed mutant CDC25B. After inducing gene amplification in the presence of 250 nM methotrexate, mutant CDC25B sequence was removed by …

Glycoengineering Of Chinese Hamster Ovary Cell For Modulating Glycoprotein Nlinked Sialylation, Chengyu Chung

Cell Culture Engineering XV

Erythropoietin (EPO), a highly glycosylated protein was widely used in anemia of kidney failure. It has been confirmed that enhancing sialic content on EPO can prolong its circulatory half-life, improving EPO therapeutic efficacy. On the other hand, limiting EPO sialylation can be beneficial in medical and diagnostic application. EPO has been evaluated as a potential radiolabeled diagnostic candidate. Thus, the development of genetic engineering strategies to finetune the sialic acid content on EPO as well other protein therapeutics is needed.

To enhance sialylation, we focused on engineering Chinese Hamster Ovary cell (CHO) to overexpress Beta-galactoside alpha-2,6-sialyltransferase 1 (ST6GAL1). Combinantorial overexpression …

Insight Into Single Cell Cloning In Serum-Free Media, Tsuyoshi Yamaguchi, Yumi Sugita, Mie Fukuda, Keina Yamaguchi, Yuka Mashio, Koichi Yamamoto, Toshiyuki Suzawa

Cell Culture Engineering XV

Chinese hamster ovary (CHO) cells have been used as host cells for the manufacturing of therapeutic recombinant proteins over the past decade. It is thought that the development of high performance cell lines, which satisfy both productivity and regulatory expectations, is one of the key success drivers to establish good manufacturing processes. The cell line for the clinical and commercial productions should be derived from a single progenitor or clone, and so the single cell cloning is an essential step during the cell line development. Recently serum-free media have been widely applied for this step. But under such conditions, the …