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Key Sessions

Nicole Borth

KEYNOTE: Genome Scale Science For CHO: From Chasing The High Productivity Miracle Gene To Exquisite Phenotype Control

BOKU University and ACIB

Nicolas Mermod

Engineering Strategies to Suppress Viral Particle Release from CHO Cells

University of Lausanne, Switzerland

08:20 - 08:30 10 mins
Chairperson's Opening Remarks
08:30 - 09:00 30 mins
Info
New Technologies: Cell Line and Host Cell Engineering
KEYNOTE: Genome Scale Science For CHO: From Chasing The High Productivity Miracle Gene To Exquisite Phenotype Control
  • Nicole Borth - Professor, BOKU University and ACIB

Traditionally in cell line development and engineering, there was a quest for individual genes which, if overexpressed or knocked out, would enhance performance, with very limited success. However, high level performance is more likely to be the result of the perfect combination of expression level of multiple genes. Today, with detailed knowledge of genomes, transcriptomes and the regulatory mechanisms that define the later, and with new tools available to manipulate these, the exquisite control of phenotypes starts to become a realistic target.

09:00 - 09:30 30 mins
Info
New Technologies: Cell Line and Host Cell Engineering
Technology Toolbox for Cell Line Development
  • Holger Laux - Fellow, Novartis Cell Line Develeopment

Chinese Hamster Ovary cells are widely used for large-scale production of recombinant biopharmaceuticals. We will present that applying transcriptomics derived approaches supported the identification of the root cause of cell growth inhibition and low productivity of a difficult to express therapeutic protein and how state of the art cell line engineering tools enabled the high expression of this therapeutic protein. Especially the combination of the recently published CHO genome with screening methods and cell line engineering tools has enabled the development of superior CHO cell lines.

09:30 - 10:00 30 mins
Info
New Technologies: Cell Line and Host Cell Engineering
CRISPR screening to improve the CHO cell host: Getting involved in the creation of the next-generation CHO line
  • Mario Pereira - Field Application Scientist, Horizon Discovery

Horizon Discovery has optimised cell-based assays which will be combined with our expertise in CRISPR screening to identify novel genetic targets that will improve the CHO cell host for biomanufacturing. We will describe the outcomes of our proof of concept work and how we intend to partner with companies interested in being at the forefront of innovation in this field.

10:00 - 10:50 50 mins
New Technologies: Cell Line and Host Cell Engineering
Morning Coffee & Poster Tour 1
10:50 - 11:20 30 mins
Info
New Technologies: Cell Line and Host Cell Engineering
Characterizing Clone Performance: A Union of Structure and Sequencing
  • Steven Huhn - Senior Scientist, Merck

Chinese hamster ovary (CHO) cells are a common tool utilized in bioproduction and directed genome engineering of CHO cells is of great interest in order to generate robust recombinant cell lines. Until recently, this focus has been challenged by the lack of flexible, high throughput gene editing modalities, high throughput screening methods, and a complete CHO genome assembly. In this work, we utilize next-generation sequencing in conjunction with CRISPR Cas9 RNPs perform genome editing targeted at the active site of the glutamine synthetase (GS) gene. We demonstrate that in vitro generated Cas9 RNP complexes containing synthetic gRNA achieved site-specific gene editing of >80% in CHO cells without selection. Furthermore, CRISPR RNPs demonstrate no effects on cell viability and growth, in contrast to harsh cytotoxicity associated with Zinc Finger Nuclease (ZFN) mRNAs. In addition, we describe how Cas9 yields a unique spectrum of mutations distinct from ZFN mediated approaches. Last, we show how CRISPR and ZFN technologies can be applied in conjunction with the TIDE (Tracking of Indels by Decomposition) algorithm in order to identify edited clonal cell populations in as little as one day, leading to high throughput screening and identification of CHO clones.

11:20 - 11:50 30 mins
Info
CRISPR and Genome Editing Technologies
Engineering Strategies to Suppress Viral Particle Release from CHO Cells
  • Nicolas Mermod - Professor, Director, Institute of Biotechnology, University of Lausanne, Switzerland

CHO cells are known to express endogenous viral elements embedded in their genome, and to release retroviral-like particles in the culture supernatant. This complicates the detection of potential contamination by viral adventitious agents, and, despite the lack of evidence of infectivity of these particles, raises safety and regulatory concerns. Using Next generation sequencing approaches, we characterized several families of endogenous retroviral elements (ERVs) present in CHO-K1 cell genome. We focused on one highly conserved ERV group of the Gammaretrovirus gender, as it was potentially functional. Transcriptome and viral particle analysis validated the functionality of ERVs from this group, and it further indicated that the mRNA and viral genome may be expressed from few (approximately 3) ERV sequences. Using CRISPR-Cas9-mediated CHO genome engineering, we mutagenized the conserved ERV sequence group. Comparison of genomic and viral particle sequences allowed the identification of one ERV that encodes the viral genome of corresponding retroviral particles. We show that particular mutations within this ERV suffice to decrease the release of viral genome-loaded particles below detection limits.

11:50 - 12:05 15 mins
Info
CRISPR and Genome Editing Technologies
Addressing Large-Scale Manufacturing of Clinical Grade Viral Vectors Using an Optimized PEI-based Transfection Process
  • Alengo Nyamay’antu - Scientific Support & Communication Specialist, Polyplus-transfection

With the progress in developing new viral vector systems guided by safety, specificity and potency considerations, several gene and cell-based therapies are now more than ever closer to being clinically approved and commercially available to treat genetic diseases.

Viral vector delivery systems, of which mainly adeno-associated viruses (AAV) and lentiviruses are produced by transient transfection of mammalian producer HEK-293 cell lines. Virus vector production using the right transient transfection method is crucial to provide the flexibility and reproducibility that is needed to scale-up from initial process development to manufacturing of high-quality grade viral vectors.

Here, we describe an optimized PEI-based virus production process for high-yielding viral vector production, compatible with different cell culture adherent and suspension systems. We further demonstrate the robust viral vector production yields, as well as the adaptability and reliability of the PEI-based transient gene expression approach to efficiently manufacture GMP-grade viral vectors at a sufficiently large scale for more advanced clinical trials, and in fine to drive commercialization of therapeutic vectors.

12:05 - 12:20 15 mins
Info
CRISPR and Genome Editing Technologies
Whole-genome sequencing-based methods to characterize cell lines used for therapeutic protein production and to determine clonality
  • Alexandre Kuhn, PhD - Director, Whole-Genome Sequencin, Selexis

Next-generation sequencing (NGS) and other novel genomic techniques represent unprecedented opportunities to improve the genetic characterization of cell lines used for therapeutic protein production. Over the past years, we have developed and validated NGS-based methods to identify integration sites, copy number and integrity of the expression construct in CHO cell lines. Recently, we have introduced a new, formal statistical test of clonality based on the fixation of single nucleotide variant mutations. We will present two approaches for direct, genomic assessment of clonality in conditions typical of cell line development and we will show how they can provide new levels of safety and efficiency for biotechnological therapeutic protein production.

12:20 - 13:35 75 mins
CRISPR and Genome Editing Technologies
Lunch and Live Labs
13:35 - 14:05 30 mins
Info
CRISPR and Genome Editing Technologies
Designing an Artificial Golgi Reactor as An Alternative to Cell Line Engineering
  • Elli Makrydaki - Graduate Student, Dept of Chemical Engineering, Imperial College London

The dominating method in producing therapeutically relevant proteins such as mAbs with the desired glycosylation profile is the traditional cell-line engineering of host cells such as mammalian cells. However, the lack of strict control can lead to increased product heterogeneity. We propose an artificial Golgi reactor for in vitro glycosylation where by immobilizing selected glycosyltransferases we aim to achieve targeted glycosylation with enhanced product quality.

14:05 - 14:35 30 mins
Info
Omics and Big Data Integration in Cell Line Development and Engineering
Structural and epitranscriptomic manipulation of RNA to enhance transgene expression
  • Niall Barron, PhD - Professor of Biochemical Engineering, University College Dublin and Principle Investigator, NIBRT

The translation of circular mRNA represents an interesting possibility for improving recombinant protein production. Initiation and termination are two rate limiting steps of translation therefore by encoding a gene lacking a stop codon on a circular RNA molecule we generated an infinite EPO open reading frame and demonstrate the production of secreted protein from a circular mRNA in mammalian cells. In addition the use of an epitranscriptomic approach to improving gene expression will be presented.

14:35 - 15:05 30 mins
Info
Systems and Synthetic Biology Applications
Putting The Horse Before The Cart: Designing Gene Expression Constructs For CHO Cell Engineering
  • Adam Brown, Ph.D. - Lecturer of DNA Engineering, University of Sheffield

Manufacture of non-natural, engineered protein formats will require purpose-built designer cell factories. While a great deal of effort has been devoted to identifying target genes for CHO cell engineering, relatively little has been spent figuring out to optimally express them. This presentation discusses design rules for creating plasmid vectors encoding complex gene combinations, describing how genetic component compositions can be designed and configured in order to optimize the performance of engineered cell factories.

15:05 - 15:35 30 mins
Info
Systems and Synthetic Biology Applications
ambr: systems enabling high throughput biologics development for intensified processes
  • Ian Ransome - Head of Product Management, ambr systems, Sartorius Stedim Biotech

Intensified processing offers many advantages to those developing biotherapeutics.  However, high throughput tools and techniques to expedite the development of cell lines, media and processes have been lacking.

Current approaches are throughput-limited by infrastructure that requires significant operational input, laboratory space and capital investment.  In this update, novel data derived from ambr 15 cell culture perfusion mimics and the ambr 250 high throughput platform will be presented.

15:35 - 16:20 45 mins
Info
Systems and Synthetic Biology Applications
Afternoon Coffee and Poster Tour 2

Channel One:

Poster 4: 15:40

CRISPR/CAS9 MULTIPLEXING OF CHINESE HAMSTER OVARY B4GAL-T1, 2, 3 AND 4 TAILORS N-GLYCAN PROFILES OF MAB AND TOTAL SECRETED HOST CELL PROTEIN

Thomas Amann, Technical University of Denmark

Poster 5: 15:50

THE CHALLENGE OF EMPLOYING HIGH-THROUGHPUT METHODS FOR PROCESS CHARACTERIZATION DURING LATE STAGE DEVELOPMENT OF BIOPHARMACEUTICALS

Susanne Ulrich, Sanofi-Aventis Deutschland GmbH, Germany 

Poster 6: 16:00

A POSTER BY APPLIKON


Channel Two:

Poster 58: 15:40

CHARACTERIZATION OF CAPTO ADHERE RESIN TO ADDRESS NEW PURIFICATION CHALLENGES FOR MONOCLONAL ANTIBODY PRODUCTION

Julia Sieben, Merck Corsier-sur-Vevey, Switzerland

Poster 59: 15:50

MAGNETIC BEAD PURIFICATION OF ANTIBODIES DIRECTLY FROM NON-CLARIFIED CELL HARVEST AT PILOT-SCALE

Nils Brechmann, PhD, KTH, Sweden

Poster 60: 16:00

ENGINEERING CHARACTERISATION OF SINGLE-USE BIOREACTORS

Ruchika Bandekar, MedImmune

16:20 - 16:50 30 mins
Info
Systems and Synthetic Biology Applications
The Relevance Of Cell Size In A CHO Fed Batch Process: Metabolic And Transcriptomic Characterization
  • Dirk E. Martens - Associate Professor, Bioprocess Engineering, Wageningen University

In a fed-batch process, using a commercially available media system a switch is observed from a cell proliferation phase to a phase where cell division is arrested and cell growth continues in the form of a threefold increase in cell size and dry weight. Metabolic flux and transcriptome analysis is applied to better understand the biological mechanisms associated with this switch.

16:50 - 17:20 30 mins
Info
Systems and Synthetic Biology Applications
Panel Discussion: Integrating New Engineering and Cell Line Development Technologies into Established Workflows
  • What new technologies have been adopted by industry and used routinely in cell line development?
  • Success rates and realities of engineering tools? e.g. genomics, genome editing, CRISPR, NGS, targeted integration
  • What is the industry status on CRISPR adoption?
  • Are titres and timelines better with new technologies compared to standard approaches?
  • Specific site integration vs. random integration: What is the impact on expression level and expression stability?
  • What are the best, efficient and smart ways to use cell line engineering in a workflow?
  • Where and how are industry incorporating new technologies and engineering for clinical and pipeline products when timelines are critical?
  • Feasibility of cell line engineering: Are people doing customised cell line engineering for individual projects and is this feasible?
  • Industry opinions on engineering: Engineer the host or look for replacements?
17:20 - 17:25 5 mins
End of Cell Line Development & Engineering 2019
08:20 - 08:30

Chairperson's Opening Remarks

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17:20 - 17:25
End of Cell Line Development & Engineering 2019

End of Cell Line Development & Engineering 2019

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