Animal Cell Cultures - Expression and Engineering

9. Danish Conference on Biotechnology and Molecular Biology May 22-23, 2014 Hotel Munkebjerg, Vejle

Front page image http://scitechdaily.com/microfluidic-system-precisely-measures-mammalian-cell-growth-rates/

9. Danish Conference on Biotechnology and Molecular Biology May 22-23, 2014 Hotel Munkebjerg, Vejle

Animal Cell Cultures - Expression and Engineering

Over the last fifty years research in cell biotechnology has revealed substantial potentials for the production of bioactive proteins and use of the cells themselves particularly in medical applications. The development of efficient and safe processes for production of novel pharmaceuticals is of significant industrial importance and subject to extensive research efforts. The increasing availability of genome editing tools and genome sequences of mammalian cell factories, such as the Chinese hamster ovary cell, and other animal cell cultures enables for the first time a systems biotechnology driven approach to cell factory design. The conference will focus on

- Perspectives in Animal Cell Factory Research - Expression and production systems - Technology platforms - Systems biology - Post translational modifications - Mammalian Cell Factories - Insect Cell Factories - Commercial perspectives and regulatory issues

The conference includes a poster session covering a broad range of topics on biotechnology as well as a commercial exhibition of equipment, consumables and services to Danish biotechnology.

Animal Cell Cultures - Expression and Engineering

Organisers Danish Biotechnological Society (DBS) is a scientific society established in 2006 in collaboration between Danish Biotechnology Forum and The Danish Society for Biochemistry and Molecular Biology. DBS is organised as part of The Danish Society of Engineers, IDA. The purpose of DBS is to facilitate networking within the field of biotechnology in Denmark, creating links between universities, research institutions, hospitals and companies. DBS represents professionals working with biotechnology and seeks to promote and communicate important issues on biotechnology, biochemistry and molecular biology to the public. DBS organises scientific conferences and meetings on biotechnology.

Working group members - Anders Vagnø Pedersen, ALK-Abelló A/S - Catharina Stenholm, Novozymes A/S - Inge Kjærbølling, Society for Biological Engineering at Technical University of Denmark - Holti Kellezi, Synapse, University of Copenhagen - Jens Gram, CMC Biologics - Jochen Förster, Technical University of Denmark - Lars Haastrup Pedersen, Aalborg University - Leif Schauser, CLC Bio - Nils Joakim Færgeman, University of Southern Denmark - Steen Gammeltoft, Glostrup Hospital - Stephanie Mesker, Technical University of Denmark - Thomas Schou Larsen, Biopeople – Innovation - Uffe Hasbro Mortensen, Technical University of Denmark

Co-organisers

The conference is organised in collaboration with Danish Society of Biochemistry and Molecular Biology as well as BioPeople

Secretariat

Birgitte Magnér-Egeberg, IDA Netværkscenter, Kalvebod Brygge 31-33, DK-1780 Kbh. V. Tel: 33 18 46 46, e- mail: [email protected]

Program

Animal Cell Cultures - Expression and Engineering

May 22 9.00 – 9.50 Registration, coffee and tea 9.50 – 10.00 Welcome by Lars Haastrup Pedersen, DBS, DK 10.00 – 11.00 Session 1 Perspectives in Animal Cell Factory Research chair: Lars Haastrup Pedersen, Aalborg University, DK 10.00 – 10.30 The genome area of CHO: from blackbox optimisation to designer cell lines? L1.1 Nicole Borth, University of Natural Resources and Life Sciences, AUT 10.30 – 11.00 Microparticles as cell-to-cell communicators to empower therapies and technology: L1.2 the case of megakaryocytic micro particles Terry Papoutsakis, University of Delaware, USA 11.00 – 13.00 Posters and exhibition, meet the speakers, and lunch 13.00 – 14.00 Session 2 Cell engineering and post-transcriptional mechanisms chair:, Thomas Schou Larsen, BioPeople, DK 13.00 – 13.30 Screening of small RNAs for impact on apoptosis and protein expression in cell cultures L2.1 Michael Betenbaugh, Johns Hopkins University ,USA 13.30 – 14.00 Efficient precise glycoengineering of cellular post translational modification pathways L2.2 Eric Paul Bennett, Copenhagen University, DK 14.00 – 15.00 Posters and exhibition, meet the speakers, hotel check in 15.00 – 16.00 Session 3 Expression and Production Systems chair: Jens Gram, CMC Biologics, DK 15.00 –15.30 The cultural divide: exponential growth in classical 2D and metabolic equilibrium in 3D environments Jonathan D. Chesnut, Thermo Fisher Scientific, USA L3.1

15.30 – 16.00 Accelerating Genome Editing in CHO Cells using CRISPR CAS9 L3.2 Helene Faustrup Kildegård, Technical University of Denmark, DK 16.00 – 17.00 Posters and exhibition, meet the speakers 17.00 – 18.00 Session 3 cont. Expression and production in CHO cells chair: Leif Schauser, CLC Bio, DK 17.00 – 17.30 CHO cell engineering for improved therapeutic protein production L3.3 Gyun Min Lee, KAIST, KOR 17.30 – 18.00 Large-scale transient gene expression platforms using HEK293 and CHO cells for protein production L3.4 Yves Durocher, Animal Cell Technology Research Group, National Research Council, CAN 18.00 – 19.00 Posters and exhibition, meet the speakers 19.30 – Conference dinner

May 23 8.30 – 9.30 Session 4 Insect Cell Factories chair: Leif Schauser, CLC Bio, DK 8.30 – 9.00 Development of Drosophila S2 insect-cell based Malaria Vaccine production processes L4.1 Wian de Jongh, ExpreS2ion Biotechnologies, DK 9.00 – 9.30 The state-of-play of baculovirus-mediated protein expression in insect cells L4.2 Svend Kjær, London Research Institute, UK 9.30 – 10.30 Posters and exhibition, meet the speakers, hotel check out 10.30 – 11.45 Session 5 Mammalian Cell Culture Production Systems chair: Nils Færgeman, University of Southern Denmark, DK 10.30 – 10.55 Industrial production systems L5.1 Jens Gram, CMC Biologics, DK 10.55 – 11.20 Cell cultures on chip - possibilities and challenges L5.2 Martin Dufva, Department of Micro- and Nanotechnology, Technical University of Denmark, DK 11.20 – 11.45 Bioprocess cost modeling guiding early stage process development L5.3 Kai M. Touw, Crucell, NL 11.45 – 13.15 Lunch, posters and exhibition, meet the speakers 13.15 – 14.05 Session 6 Regulatory Issues and Commercial Perspectives chair: Thomas Schou Larsen, BioPeople, DK 13.15 – 13.40 Regulatory Environment of White Biotechnology with a Shade of Red L6.1 Carsten Hjort, Novozymes A/S, DK 13.40 – 14.05 Biological engineering – a stronghold in Danish biotech L6.2 Martin Bonde, Dansk Biotek 14.05 – 14.40 Posters and exhibition, meet the speakers 14.40 – 15.55 Session 7 Mammalian Cell Factories and Systems biology chair: Mikael Rørdam Andersen, Technical University of Denmark, DK 14.40 – 15.05 Engineering Biological Networks in CHO Cell Lines L7.1 Mikael Rørdam Andersen, Department of Systems Biology, Technical University of Denmark, DK 15.05 – 15.30 In-vivo like performance of microgravity spheroids in-vitro L7.2 Stephen J. Fey, University of Southern Denmark, DK 15.30 – 15.55 Towards Holistic Biotechnology – L7.3 An Industrial Perspective on Next Generation Cell Culture Processes Ali Kazemi Seresht Cell Culture Technology, Novo Nordisk A/S, DK 15.55 – 16.00 Closing remarks Lars Haastrup Pedersen, DBS, DK

Animal Cell Cultures - Expression and Engineering

Lecture abstracts

Animal Cell Cultures - Expression and Engineering

L1.1 The genome area of CHO: from blackbox optimisation to designer cell lines?

Nicole Borth

University of Natural Resources and Life Sciences Vienna (BOKU) and Austrian Center of Industrial Biotechnology (ACIB), Austria

During the last 25 years, recombinant protein production in Chinese Hamster Ovary cells has reached a level of optimisation that has turned it into an “established technology”. Indeed, the improvements in yields achieved are remarkable: titers have increased from 0.1-0.4 g/l to reliable 2-6 g/l, with top values of 20 g/l reported. Nevertheless this achievement was based on empirical approaches and was largely due to enhancements of the medium composition and the higher cell densities thus reached. Details of what happened inside the cells have remained mostly a blackbox, despite the advances accomplished. With the availability of a genomic sequence, both of several CHO cell lines and the reference genome from the Chinese hamster, there now is the opportunity to accomplish a step change in our understanding and control of both the bioprocesses and cell behaviour. In parallel, new tools and methods are emerging that enable precise genome and epigenome editing, which together with the sequence information will enable the generation of designer cell lines, with precisely edited properties for different product categories.

The present talk will give an overview over the available CHO databases and sequences and will then focus on how this genomic information can be used to obtain a comprehensive understanding of the regulatory network in place in this industrially used production cell line. In addition, the currently discussed plans for development of infrastructure and software to be available at www.CHOgenome.org will be discussed.

Animal Cell Cultures - Expression and Engineering

L1.2 Microparticles as cell-to-cell communicators to empower therapies and technology: the case of megakaryocytic microparticles

Eleftherios Terry Papoutsakis & Jinlin Jiang

University of Delaware, USA

A long-standing goal in cell-culture technologies is the ability to produce human blood cells for transfusion medicine. Another important goal is to develop robust differentiation technologies of stem cells, technologies that could be transferred to the clinic but also used in in vitro investigational experimentation. Among blood cells, platelets, needed for blood coagulation and vascular repair, are an expensive “product” in limited supply. Production of platelets in a “blood factory” is recognized as a grand challenge that remains elusive. Platelets derive from polyploid megakaryocytes (Mks) in the bone marrow and lung vasculature, under biomechanical forces. We will show how important these forces are for producing functional platelets and their precursors, as well as small, anuclear particles, Mk microparticles (MkMPs). MkMP generation was dramatically enhanced (up to 47 fold) by shear flow. Significantly, co-culture of MkMPs with hematopoietic stem and progenitor cells (HSPCs) promoted HSPC differentiation to Mks without exogenous thrombopoietin, thus identifying, for the first time, a novel and previously unexplored potential physiological role for MkMPs. This demonstrates the extraordinary ability of these MkMPs in programming HSPCs. We will discuss our efforts to understand the mechanisms by which MkMPs target and act upon cells. How general is the production of MPs? Most cells release into the extracellular environment these very small particles (typically less than 1 micron) known either as microvesicles or MPs. MPs shed from the cell surface upon activation or some other mechanism, here under shear flow. They derive from the endosomal compartment after fusion with the plasma membrane, and are increasingly recognized as important players in intercellular communication by transferring proteins, lipids, RNA, and perhaps DNA, between cells. They do so with good target specificity and thus, one can argue for producing and using them for regenerative-medicine applications, as well as in experimental investigations to deliver “cargo” to specific cell types. So, not proteins, not cells, but rather active biological entities posing new production, formulation and delivery challenges in the development of new cell-culture based technologies for human therapy and basic research.

L2.1 Screening of small RNAs for impact on apoptosis and protein expression in cell cultures

Michael Betenbaugh

Johns Hopkins University ,USA

Mammalian cells are responsible for the production of the majority of biopharmaceuticals produced in biotechnology today. One of the central cellular sub-processes of mammalian cell culture is programmed cell death or apoptosis. This presentation will provide several different microRNA approaches underway in our laboratory to examine and control apoptosis and protein production in mammalian cultures. The activation of apoptosis in biotechnology can shorten the production cycle for cells in culture and lower viabilities and yields of biopharmaceuticals. In addition, the disregulation of apoptosis is a key activator of cancer and the activation of the programmed cell death cascade is a major approach for cancer therapy. Given the central role of apoptosis in mammalian cell culture processing, our group is undertaking a systems and omics analysis of the apoptosis cascade in an attempt to both control its activation and link its existence to other cellular processes. In one approach, we are elucidating the role of microRNAs in the apoptosis cascade. The ability to decipher microRNA’s involvement in apoptosis is being used to limit program cell death for biopharmaceutical applications and likewise to design potential cancer treatment strategies. In another application, we are examining the capability for microRNAs to alter production of target proteins of interest. Finally we will explore potential links between apoptosis and cellular metabolism in order to understand how these processes may be related and important for dictating the cellular responses to external stresses. A systems approach to important physiological processes such as apoptosis processing will enhance our ability to control mammalian cellular events and in turn lead to improved production of biopharmaceuticals and new methodologies for the treatment of disease.

Animal Cell Cultures - Expression and Engineering

L2.2 Control of protein glycosylation in Chinese Hamster Ovary (CHO) cells

Yang Zhang, Yoshiki Narimatsu, Adnan Halim, Sergey Vakhrushev, Henrik Clausen, Eric Paul Bennett

University of Copenhagen, Denmark.

Glycosylation is the most abundant and diverse posttranslational modification of proteins, and glycosylation directs important biological functions for proteins. Several types of protein glycosylation can be predicted with reasonable reliability by the protein sequence context (e.g. N-linked, O-Fuc, O-Glc) and substantial knowledge of these glycoproteomes is available. In contrast, several types of O-glycosylation (e.g. O-GalNAc, O-Man, O-GlcNAc) remain more elusive and simple sequence context motifs are not useful for prediction of glycosites. This in combination with great heterogeneity of O-glycan structures at individual glycosites has hampered our knowledge of these types of glycosylation as well as the diverse biological functions they serve. We have started employing precise gene editing technologies (ZFNs, TALENs, CRISPR/Cas9) to engineer cell systems with simpler and more homogenous glycosylation by knocking out or knocking in key glycosyltransferase genes, which enables lectin enrichment and proteome- wide discovery of O-glycosites using “bottom-up” mass spectrometric analysis (1). We have implemented this on O-GalNAc and O-Man glycoproteomes of human cell lines from different organs (2,3), and we are probing the contribution of individual isoenzymes involved in initiation of O-glycosylation using comparative analysis of glycoproteomes (4). These studies are generating surprising new insight into the abundance of O-glycosylation and new biological functions. This presentation will present our glycoproteome studies of CHO cells and highlight examples of new important biological functions of site- specific O-glycosylation.

Keywords: glycoproteomics, glycoproteins, post translational modifications

1. Steentoft, C., Vakhrushev, S. Y., Vester-Christensen, M. B., Schjoldager, K. T., Kong, Y., Bennett, E. P., Mandel, U., Wandall, H., Levery, S. B., and Clausen, H. (2011) Nat Meth 8, 977-982

2. Steentoft, C., Vakhrushev, S. Y., Joshi, H. J., Kong, Y., Vester-Christensen, M. B., Schjoldager, K. T., Lavrsen, K., Dabelsteen, S., Pedersen, N. B., Marcos-Silva, L., Gupta, R., Paul Bennett, E., Mandel, U., Brunak, S., Wandall, H. H., Levery, S. B., and Clausen, H. (2013) EMBO J 32, 1478-1488

3. Vester-Christensen, M. B., Halim, A., Joshi, H. J., Steentoft, C., Bennett, E. P., Levery, S. B., Vakhrushev, S. Y., and Clausen, H. (2013) Proc Natl Acad Sci USA 110, 21018-23

4. Schjoldager, K. T., Vakhrushev, S. Y., Kong, Y., Steentoft, C., Nudelman, A. S., Pedersen, N. B., Wandall, H. H., Mandel, U., Bennett, E. P., Levery, S. B., and Clausen, H. (2012) Proc Natl Acad Sci USA 109, 9893-98

L3.1 Simple and Versatile Genome Editing Tools for Large scale Cell Engineering

Jonathan D. Chesnut

Life Sciences Solutions Group, Thermo Fisher Scientific, USA

Recent advances in sequencing, bioinformatics and genomic editing have changed how we approach development of new mammalian cell platforms. For instance, deeper knowledge of the genome is allowing identification and targeting of specific genes for modification that will lead to new cell platforms such as bioproduction line with optimized glycosylation and minimal proviral sequences or specifically edited pluripotent cells that could be used as disease models.

The newest in a long line of editing tools, Clustered Regularly Interspaced short palindromic repeat (CRISPR), is a bacterial adaptive immune system from Streptococcus pyogenes that has been recently demonstrated to help rapidly generate engineered cell lines, model organisms, and perform large scale gene modifications in a wide variety of hosts. Due to its simple design including Cas9 nuclease and a non- coding guide RNA (gRNA) with target specificity defined by only a short 17-21 base nucleotide region, this system is an attractive tool for large scale genome engineering. Described here are sets of versatile CRISPR delivery formats that address both small and large-scale genome editing needs in a wide range of host and cell types including stem cells. This work includes a combination of ready to transfect formats that circumvent the need for host specific promoters and reduce the hands-on time needed for generating target specific CRISPR systems. It also allows the user to edit multiple loci or alleles simultaneously. The simple and easy to design tools described here hold great promise in large-scale genome and cell engineering applications.

Animal Cell Cultures - Expression and Engineering

L3.2 Accelerating Genome Editing in CHO Cells using CRISPR CAS9

Carlotta Ronda1,#, Lasse Ebdrup Pedersen1,#, Henning Gram Hansen1, Thomas Beuchert Kallehauge1, Michael J. Betenbaugh1,2, Alex Toftgaard Nielsen1 and Helene Faustrup Kildegaard1

1The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark.

2 Johns Hopkins University, USA

# These authors contributed equally to the work

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, we demonstrate the efficacy of the CRISPR Cas9 technology in CHO cells by generating site-specific gene disruptions in COSMC and FUT8, which encode proteins involved in glycosylation.

A CHO codon-optimized Cas9 was applied together with sgRNAs against COSMC and FUT8 to modify the genome of CHO K1 cells. FUT8 knockout cells were selected by supplementing media with Lens culinaris agglutinin (LCA) and double knockout cells were identified by cell staining. The efficiency of the CRISPR Cas9 system and the generated indels was further analyzed by T7 endonuclease assay, topo TA cloning and deep sequencing.

The tested single guide RNAs (sgRNAs) created an indel frequency up to 47.3% in COSMC, while an indel frequency up to 99.7% in FUT8 was achieved by applying lectin selection. All eight sgRNAs examined in this study resulted in relatively high indel frequencies, demonstrating that the Cas9 system is a robust and efficient genome-editing methodology in CHO cells. Deep sequencing revealed that 85% of the indels created by Cas9 resulted in frameshift mutations at the target sites, with a strong preference for single base indels. The proven functionality of the CRISPR Cas9 system to edit CHO genomes has the potential to accelerate genome editing and synthetic biology efforts in CHO cells.

L3.3 CHO Cell Engineering for Improved Therapeutic Protein Production

Gyun Min Lee

Department of Biological Sciences, KAIST , Republic of Korea

CHO cells are widely used for the production of therapeutic protein due to their ability to synthesize, fold, glycosylate, and secrete complex proteins in large-scale suspension culture. CHO cell engineering has been targeted mainly to increase the specific productivity and/or the time integral of viable cell concentration. Various strategies such as the overexpression of anti-apoptotic genes and pro-autophagic genes, overexpression of chaperones, and overexpression of proprotein convertases, eventually leading to improved protein production, will be discussed. In addition, the strategy to overcome the accumulation of toxic wastes such as lactate and ammonia will be discussed.

Animal Cell Cultures - Expression and Engineering

L3.4 Large-scale transient gene expression platforms using HEK293 and CHO cells for protein production

Yves Durocher

Life Sciences | NRC Human Health Therapeutics Portfolio National Research Council Canada, Montreal (QC), Canada

Large-scale transfection of HEK293 cells is a highly valuable tool for the fast generation of mgs to grams quantities of r-proteins required for R&D purposes. More recently, new CHO-based transient expression platforms have been developed with some success, even though productivity is often lower compared to HEK293 cells. I will present data describing our 293 and CHO platforms for the production of various proteins. For more difficult to express proteins, or for proteins needed in large quantities, we also developed a CHO pool platform that allows the generation of stable pools high levels of monoclonal antibodies in less than 5 weeks post-transfection. This platform can also be used to derive stable cell lines for manufacturing therapeutic r-proteins candidates.

L4.1 Development of Drosophila S2 insect-cell based Malaria Vaccine production processes

Wian de Jongh

ExpreS2ion Biotechnologies, Denmark

Drosophila S2 insect cell expression is less known than the extensively used Spodoptera or Trichoplusia ni (Hi-5) insect cell based Baculovirus expression system (BEVS). Nevertheless it has been used in research for almost 40 years. The cell line was derived from late stage Drosophila melanogaster (Fruit fly) embryos by Schneider in the 1970s, who named the cell line Drosophila Schneider line 2 (synonyms: S2, SL2, D.mel. 2). The system has been widely applied to fundamental research, where the availability of the whole genome sequence of Drosophila melanogaster (1, 2) and the S2 cells’ susceptibility to RNA interference methods (3, 4) have enabled genome wide RNAi screening and whole genome expression analysis techniques to be used to great effect. S2 cells have proved to be highly effective for the production of proteins from a great variety of protein classes (5), such as: viral proteins, toxins, membrane proteins, enzyme, etc. Recent publications have also shown the strength of the S2 system in expression of Virus Like Particles (VLPs) (6).

ExpreS2ion has developed the ExpreS2, Drosophila S2 platform to achieve improved yields for difficult to express proteins. Furthermore, several technologies have been developed to improve the ease of use of the system, as well as enable fast and efficient screening of multiple constructs. For example: An efficient transient transfection screening method using suspension cultures will be presented. This method can be applied to 1ml volumes in 96-well deep-well plates or larger volumes in shake flasks. Examples of a range of proteins expressed in the system will be presented, including immune modulatory proteins (IDO and PD- L1), secreted heterodimers, malaria antigens (Rh5 and VAR2CSA), and VLPs (HIV GAG).

The Drosophila S2 expression system has been used for antigen manufacture up to Phase II clinical trials. Currently, ExpreS2ion is developing S2 based production processes for two malaria vaccine clinical trails with Oxford University (Rh5) and Copenhagen University (VAR2CSA). The system is well suited to both R&D and clinical development, with particular advantages for difficult to express secreted proteins.

(1) Adams M.D. et al. Science 2000 287:2185-2195

(2) Ashburner M, et al. Genome Res. 2005 Dec;15(12):1661-7

(3) Neumüller RA, et al. Wiley Interdiscip Rev Syst Biol Med. 2011 Jul-Aug; 3(4):471-8

(4) D’Ambrosio M.V. et al. J. Cell Biol. Vol. 191 No. 3 471–478

(5) Schetz J.A. et al. Protein Expression in the Drosophila Schneider 2 Cell System, Current Protocols in Neuroscience, 2004

(6) Yang. L. et al. J Virol. 2012, Jul;86(14):7662-76.

Animal Cell Cultures - Expression and Engineering

L4.2 The state-of-play of baculovirus-mediated protein expression in insect cells

Annabel Borg, Roger George, Sara Kisakye-Nambozo, Svend Kjær

Protein Purification Facility, London Research Institute, United Kingdom

The Protein Purification Facility (PPF) at the LRI supports research labs with expertise in an array of technologies related to protein expression, purification and characterization (such as affinity determination, Multi-Angle Laser Light Scattering and crystallization).

To accommodate the expression of a wide variety of target proteins (intra/extracellular, single protein/protein complexes, post-translationally modified) that are submitted to the PPF, the versatile baculovirus-mediated technology was adapted and has currently been used for more than 5 years to express hundreds of proteins.

We here present a review of the baculovirus technology with emphasis on recent developments and present our set-up with examples of expression of single proteins as well as protein complexes using the MultiBac technology. Moreover, we will discuss means of scaling-up and yield optimization.

L5.1 Industrial Production systems

Jens Gram

CMC Biologics, Denmark

Keywords: Biologics/Biopharmaceuticals, Processing, Single Use, CMO, CMC

The technologies involved in the manufacture of Biologics* are currently undergoing a rapid development.

This presentation reviews current approaches and requirements to:

 Processing technologies and batch sizes  Processing facilities and equipment capabilities and capacities  Expression systems from a Biologics* Contract Manufacturers’ point of view.

The foundation for and future outlook for some important developments including the implementation of single use technologies and the associated challenges are discussed.

*Biologics: Drug Products, containing biological molecules produced in living organisms as active pharmaceutical ingredients – Another term often used is “Biopharmaceuticals”.

Animal Cell Cultures - Expression and Engineering

L5.2 Cell culture chips - opportunities and challenges

Martin Dufva

Technical University of Denmark, Denmark

Mammalian cell culture in miniaturised microfluidics chips has large potential in numerous applications. We have developed a microfluidics platform to which we can interface various chips that simulates simple or more advanced multicellular models of human physiological compartments. The simplest chip is a straight channel (area of 6 mm^2) and has been used to study basic transport phenomena during perfusion and cell culture. From these studies it was clear that extensive paracrine and autocrine signalling governs stem cell differentiation. Applied correctly, flow based differentiation improved differentiation significantly. Other chips are used to create biological barriers and are used to study migration in 3D and drug penetration. These chips simulates for instance intestine/blood barrier by separating two individually addressable chambers with a membrane or thin gels. One chamber simulates the blood side and the other the intestine. Over the last years we have developed 3D chips for organ modelling. Our aim is to build a extra-corporal liver as life support unit for liver diseased patient. The 3D chips provide large surface to volume area and the developed fabrication method can be scalable to litre sized bioreactors. We estimate that a 0.12 cm^3 cube of our 3D chip has a functional surface area of a 75 cm^2 meaning that a dm^3 corresponds to about 750,000 cm^2 (or 7.5 m^2). The 3D chips can support stem cell differentiation into hepatocyes from human induced pluripotent stem cells. Our future perspective is to have 3D chips with similar heptocyte density as in the liver perforated by a combination of micro fabricated channels and capillaries formed using sprouting endothelial cell.

The technologies presented here can be used for many applications including animal replacement for toxicology studies, research on tissue and cell development, organ replacements and life support units but possibly also for large scale production of cells and recombinant proteins.

Acknowledgement.

This work has been and is supported by the EU project NanoBio4Trans Contract No 304842, SThis work was supported by grant no. 2106-08-0018 “ProCell,” grant no. 2106-05-0047 “BioXTAS,” and grant no. DSF- 09- 067112, under the Programme Commission on Strategic Growth Technologies; the Danish Agency for Science, Technology and Innovation; and EU FP7 grant agreement no. NMP4-SL-2008-214706 “EXCELL.”

L5.3 Bioprocess cost modeling guiding early stage processes

Kai M. Touw

Crucell, Netherlands

Cost of goods can be an important driver in process decisions. This especially holds for vaccine production as these are aimed to be low cost products in a highly competitive and/or low/mid income markets. To get a better understanding of a production process and evaluate process options, cost of goods modeling is a useful tool. Within Crucell (a Johnson & Johnson company) models of production processes are used to guide process development and assist facility design by giving insight into the cost of goods of the current process.

In the case study presented, a comparison will be made between scenarios where traditional stainless steel manufacturing is compared to a fully disposable facility. Next to this it will be shown how bioprocess modeling can be used to evaluate the CoGs impact of development efforts on medium optimization and alternative unit operations or process options.

Keywords: Process Modeling, Cost Evaluation, Vaccine Production, Process Optimization

Animal Cell Cultures - Expression and Engineering

L6.1 Changes in the regulatory environment of White Biotechnology

Carsten Hjort

Novozymes A/S, Denmark

The regulatory environment of White Biotechnology (i.e. industrial biotechnology) has historically been developed in different ways across the world, resulting in variable requirements at regional and national levels. This is contrast with Red Biotechnology (bio-pharmaceuticals), where the introduction of the ICH guidelines have provided a fairly uniform global framework.

This situation is however gradually changing, with a trend towards harmonization of the requirements on white biotechnology products at a global scale. Regulatory agencies, scientists and official expert panels talk together across continents about legal frameworks as well as scientific considerations – leading to more coherence overall. Despite this trend, though, the requirements for strain construction, strain characterization and toxicological characterization still remains quite different in the European Union, in Japan, in China, in the USA, and in some other countries.

The scientific development on microbial expression systems have changed dramatically during the last 10 years. The old regulatory requirements were implemented when microbial genomes were largely not sequenced and poorly understood. Today the genomes of most expression systems have been fully sequences and that has led to identification of “sequences of concern” such as gene clusters encoding secondary metabolites of concern, antibiotic resistance markers and other genes of concern. At Novozymes we have used this increasing level of knowledge to design new generations of host strains where such elements of concern have been deleted.

The global harmonization and the science development leads to requirements becoming more and more stringent, to some extent inspired by the guidelines adopted by the European Union for fermentation products manufactured by genetically modified microorganisms. The complexity and overall level of what is required of white biotechnology companies has become such that we need to take this fully into account at the very beginning of an industrial product research and design process.

Our industry welcomes the trend towards harmonization of requirements at a global scale. We however need regulatory agencies and expert panels to take onboard the scientific progresses accomplished in making our microorganisms increasingly well-characterized and free of safety concerns. The Safe Strain Lineage concept, developed by the industry and external scientists together, is taking advantage of the body of knowledge generated on industrial microorganisms, to substitute animal safety studies by molecular strain characterization. This concept has been promoted to regulatory agencies around the world and is presently being used in industry dossiers for the purpose of product approvals. It remains to get fully endorsed by official expert panels.

Overall, continued and constant exchanges of views between academia, the industry and authorities globally are necessary, so that industry scientists and official experts reach common understandings and agree on mutual expectations.

L6.2 Biological engineering - a stronghold in Danish biotech

Martin Bonde

CEO Epitherapeutics ApS, Denmark

Biological engineering has become a cornerstone in the production of pharmaceutical drugs. The export of medicine in 2013 was DKK 72 billion (up from DKK 40 billion in 2008) and as such by far the largest export industry in Denmark. Export of industrial enzymes was DKK 6.2 billion and as such also plays a key role in the creation of wealth in Denmark. The stronghold in biological engineering takes its foundation almost 100 years ago long before molecular biology entered the scene and became key in biological production.

Dansk Biotek, an industry organization for Danish biotech and pharma companies, was founded in 1987 with the aim of establishing a communication platform to improve the understanding of the importance of molecular biology and gene splicing. Dansk Biotek, and other supporters of the industrial use of molecular biology, succeeded in conveying the message such technology is key to produce modern biotechnological product in a safe and efficient way thereby laying the ground for the industry the way we know it today.

Looking ahead we are facing an ever increasing globalization in the biotech industry. This represents both a threat and an opportunity. The ability to access services and goods of high quality, but at only a fraction of the price in the Western world, represents an opportunity to optimize research and production and focus on core competences that are believed to be the strongest value drivers. The threat obviously is that scientists and technologies from developing countries will become highly competitive with what we have classically considered our core competences. Biological engineering is a complex and highly specialized discipline and the capabilities and competences in the field in Denmark are currently very strong due to decades of expertise building. Nevertheless, a focused, dedicated and continued research effort is necessary in order to keep the leading position in the years to come.

Animal Cell Cultures - Expression and Engineering

L7.1 Engineering Biological Networks in CHO Cell Lines

Mikael R. Andersen1, Yuzhou Fan2, Dietmar Weilguny2, Christian S. Kaas1,3, Claus Kristensen3, Daniel Ley1, Anne Mathilde Lund1, Helene F. Kildegaard1

1 Technical University of Denmark, Denmark. 2 Symphogen A/S, Denmark 3 Novo Nordisk A/S, Denmark

Despite the use of Chinese Hamster Ovary (CHO) cells for production of pharmaceutical proteins since the 1980s, the genetic characterization and engineering of these cells have been lacking behind other model cell factories, e.g. bacterial or fungal hosts for protein productions. The economic interest in the area is substantial – biopharmaceutical proteins is a >100 million USD market – but until lately, the cost of sequencing a mammalian genome has been prohibitive. However, the recent publication of the genome sequences of several CHO cell lines and a draft genome for the progenitor hamster has sparked a genomic revolution both in the academic and the industrial CHO cell community.

Activities in our lab and with collaborators have adopted biological-network-based approaches known from other organisms, and applied them to CHO cells. In this talk, I will present methodologies and findings from our study of the protein secretion pathway. Methods include data integration from a diverse set of next- generation omics technologies, including DNA sequencing (DNA-seq), RNA-seq, proteomics, and N-glycan sequencing, with reconstructed biological networks. In particular we apply protein interaction networks and metabolic networks.

Among other findings, I will demonstrate how our research has uncovered divergence at the genome and regulatory levels between CHO cells and wild type mammalian cells. Furthermore, the applied approaches have proven powerful in identifying targets for cell line engineering.

L7.2 The cultural divide: exponential growth in classical 2D and metabolic equilibrium in 3D environments

Krzysztof Wrzesinski1, Adelina Rogowska-Wrzesinska1, Kamil Borkowski2, Vasco Botelho Carvalho1, Stephen J. Fey1

1 University of Southern Denmark, Denmark. 2 University of Copenhagen, Denmark

Cellular metabolism can be considered to have two extremes: one is characterized by exponential growth (in 2D cultures) and the other by a dynamic equilibrium (in 3D cultures). We have analysed the proteome and cellular architecture at these two extremes and found that they are dramatically different. Ultrastructurally, actin organization is changed, microtubules are increased and keratins 8 and 18 decreased. Metabolically, glycolysis, fatty acid metabolism and the pentose phosphate cycle are increased while Krebs cycle and oxidative phosphorylation is unchanged. Enzymes involved in cholesterol and urea synthesis are increased underpinning the attainment of cholesterol and urea production rates seen in vivo. DNA repair enzymes are increased even though cells are predominantly in G0. Transport around the cell – along the microtubules, through the nuclear pore and in various types of vesicle has been prioritized. There are numerous coherent changes in transcription, splicing, translation, protein folding and degradation. The amount of individual proteins within complexes is shown to be highly coordinated. Typically subunits which initiate a particular function are present in increased amounts compared to other subunits of the same complex.

We have previously demonstrated that cells at dynamic equilibrium can match the physiological performance of cells in tissues in vivo (Wrzesinski and Fey 2013, Wrzesinski et al 2013, Fey and Wrzesinski 2012). Here we describe the multitude of protein changes necessary to achieve this performance.

References:

K. Wrzesinski and S.J. Fey. Toxicology Research; 2(2) 123-135; 2013

K. Wrzesinski et al. Toxicology Research, 2(3) 163-172; 2013

S.J. Fey and K. Wrzesinski. Toxicological Sciences 127(2) 403-411; 2012

Animal Cell Cultures - Expression and Engineering

L7.3 Towards Holistic Biotechnology – An Industrial Perspective on Next Generation Cell Culture Processes

Ali Kazemi Seresht

Novo Nordisk A/S, DK

The biopharmaceutical industry will need to define the biotechnology area of tomorrow as a sustainable, knowledge-driven, platform-oriented and an efficiently operating unit. Next generation cell culturing will need to be based on an API-centric platform approach, which requires a holistic mind set, flexible toolboxes, and cross-disciplinary knowledge generation. The industry may have to re-think classical bioprocessing, approach a cell physiology-oriented medium design, and move from a probabilistic-based cell line development to targeted, biologically meaningful host cell engineering.

This talk aims at highlighting challenges faced when sustainability is being compromised by high throughput, and illustrates some recent developments and trends towards re-defining cell culturing of tomorrow – from an industrial perspective.

Poster abstracts

Animal Cell Cultures - Expression and Engineering

P1 Engineering the CHO Cell

Bjørn Voldborg

Technical University of Denmark, Denmark

Over the past two decades, the CHO Cell has become increasingly used as the expression host of choice in the industrial production of therapeutic proteins. Despite this fact, the CHO cell as production host is poorly understood and most of the development within this area has been done to optimise the expression of a specific protein product as the end goal. As the three main drivers that have been driving the cell factory development in the microbial field, (1) whole genome sequences and analytical ”omics”, (2) efficient genetic modification tools and (3) genome-scale in silico models, have now become available for the CHO cells, it is now possible to adress the CHO cells as cell factories for the production of therapeutic proteins in a completely novel and more general way.

At the NNF-Center for Biosustainability (CFB), we have engaged in an 8-year project based on genome scale science to develop CHO cell lines optimised for the industrial production of therapeutic proteins. Using full scale metabolic models, cutting edge genetic engineering tools and high throughput technologies, we will systematically engineer CHO cell lines to improve expression, secretion, growth, glycosylation, metabolism, etc… to obtain a panel of optimised CHO cell lines specialised for high productivity of therapeutic proteins with custom-designed homogenous glycosylation, made for optimal performance under large scale bioprocessing conditions.

The project is organised in a 50-50 fashion into basic research, performed by 3 Scientific sections and into a Core group to translate the findings of the scientific sections into optimised CHO cell lines that are to be made accessable for the industrial production of therapeutic proteins. The Core group is working with an iterative loop approach, to generate genomically targetted engineered cell lines in high throughput format and validating the effects of the engineered traits using high throughput “omics” approaches. These findings are fed back to the Scientific sections and into the metabolic models, to generate a new set of genes to be engineered in the following rounds of the iterative loop.

Animal Cell Cultures - Expression and Engineering

P2 CHEF1® – An Efficient Platform for Generation of Production Cell Lines

Howard Clarke, Christian Müller and Poul Baad Rasmussen

CMC Biologics

CHEF1® cell line development platform creates stable, production-quality clonal CHO cell lines within an industry leading 12 week timeframe (from transfection to top clone). The CHEF1 expression system utilizes regulatory domains of the Chinese Hamster Elongation Factor 1 (CHEF1) housekeeping gene to drive stable recombinant protein expression in Chinese Hamster Ovary (CHO) cells. High titer pools are obtained post transfection and through single cell cloning only ~200 clones need to be screened for identification of high producing production cell line candidates. The high titer pools also offer the possibility to get early material produced at gram scales (mAb’s) within 5-6 weeks from transfection. A new version of our CHEF1 vector leads to clones with improved productivity and shows promise to take our CHEF platform to the next stage.

P3 CHO cell line engineering and design – protein purification and analysis

Stefan Kol and Bjørn Voldborg

Technical University of Denmark

One of the main goals of the CHO Cell Line Engineering and Design section will address the desire to obtain high yields and high quality glycoprotein targets in an optimized CHO production host. For this purpose, we will express and purify 5 different glycosylated model proteins. These proteins are Rituximab, Enbrel, erythropoietin, C1 inhibitor (SerpinG) and α1-antitrypsin (SerpinA1). To be able to select high expressing single cell clones of CHO cell lines and to systematically determine yields, we plan to develop quantification assays for all five model proteins. Currently we have the assays for Rituximab, Enbrel and erythropoietin in place. These results are summarized in this poster.

Animal Cell Cultures - Expression and Engineering

P4 CHO cell line engineering- molecular biology support.

Sara Petersen Bjørn, Patrice Menard and Bjørn Voldborg.

Technical University of Denmark, Denmark

The goal of the CHO cell line engineering project at the Novo Nordisk Foundation Center for Biosustainability is to create new and improved CHO cell lines for the production of biopharmaceuticals. The method of choice to achieve this is to introduce specific genetic modifications and assay the resulting phenotype, e.g. the effect on protein glycosylation or growth characteristics. To introduce genetic modifications we use genome editing tools like ZFNs, TALs and CRISPR/Cas9. The CHOcore molecular biology team supports the project with construction of relevant plasmids, detailed analysis of genomic modifications and purification of relevant macromolecules.

Examples of cloning platforms and analysis methods of genomic modifications will be presented.

P5 CHO Cell Line Engineering – CFB CHO Cell Biology

Johnny Arnsdorf, Karen Kathrine Brøndum, Zulfiya Sukhova, Marianne Decker and Bjørn Gunnar Voldborg.

Technical University of Denmark, Denmark

The majority of recombinant therapeutic proteins are produced by Chinese Hamster Ovary (CHO) cells. These cells are often the choice of cells for expression of therapeutics in the biopharmaceutical industry because CHO cells have the ability to perform the complex post translational modifications required for drug efficacy and stability. Nevertheless, despite development of many specific individually optimized CHO production hosts cell lines by multiple companies, development of a “Super CHO” cell line is an attractive strategy to pursue. The objective of CHO cell line engineering at CFB is to create a panel of CHO cell lines with improved multifunctional properties e.g. high recombinant protein expression, predictable glycosylation pattern, virus resistance, and optimal engineered metabolic pathways. Technologies taking the advantage of zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced palindromic repeats (CRISPR) systems are used to generate targeted knockout and knock-in these CHO cell lines. Subsequently, a number of drug model proteins are expressed in the various cell lines in order to test for the resulting phenotypic effects of the genomic modifications. Accordingly, an overview of the ongoing CHO cell line engineering activities at the CFB CHO Cell Biology unit will be presented.

Animal Cell Cultures - Expression and Engineering

P6 Cell culture related services at Bioneer A/S

Peter Ravn and Holger Riemann

Bioneer A/S, Denmark

Bioneer is an independent, research-based service company within biomedicine, biomedical technology and biotechnology. Bioneer participates in research projects and are offering services related to cell culture and expression.

Bioneer offers to produce proteins in mammalian cells and is able to handle all steps in the production process including genetic design, optimization of expression, small scale protein manufacturing (expression), and downstream processes. We carry licenses for systems for both transient gene expression and expression in stable cell lines. This enables customers to have proteins produced without the need to acquire expensive licenses.

Bioneer also performs services related to cell cultivation. Examples are assistance in development of devices for cell cultures and services which constitutes smaller elements of a protein production process.

Examples are given of services and projects, which have been carried out. These include transient gene expression for customers, protein production in stable cell lines, and assistance in development of single use bioreactors.

P7 Protein network reconstruction of CHO cell secretory pathway

A.M. Lund1, H.F. Kildegaard, C.S. Kaas, C. Kristensen, M.R. Andersen

Technical University of Denmark, Denmark 1E-mail: [email protected]

Protein secretion is one of the major bottlenecks in the productivity of recombinant protein in mammalian cells. The Chinese hamster ovary (CHO) cell-line is the predominant mammalian industrial cell line being used to produce recombinant therapeutic proteins. Approaches for improve protein expression in CHO cells has often been based on altering the expression of some of the machinery components in the secretion system with the objective to increase a particular recombinant protein. So far, there have been limited studies of the cell biology of the CHO cell and the potential of cell line engineering. To elucidate the poorly understood cellular processes that control and limit recombinant protein production and secretion, a system-wide study of CHO cells was initiated to identify possibly engineering targets relevant for therapeutic protein production.

In this study, we employ a guided approach that integrates protein interaction network, gene expression and comparative studies of mouse and CHO cells for identification of genetic targets of the secretory pathway. The complex cellular machineries of the protein secretion pathway are reconstructed employing legacy knowledge of human and mouse. The full reconstruction of the secretion pathway is then combined with a catalogue of CHO RNA-Seq gene expression data from a diverse set of growth conditions and cell lines. In parallel a similar mouse RNA-Seq gene expression data set was generated from the mouse ENCODE project.

The secretion pathway including the process of protein folding, terminally misfolded proteins, and unfolded protein response constitutes of more than 700 components in addition to several markers of ER stress, apoptosis and autophagy. A comparative expression analysis of CHO cells and mouse was conducted, where clusters of genes with similar expression patterns were identified, that allows for identification of proteins and association with changed network arrangement in CHO cells. Using the RNA-seq data, a differential gene expression analysis of the constructed CHO secretion pathway will provide a unique identification of active components to increase the productivity of recombinant proteins.

Animal Cell Cultures - Expression and Engineering

P8 High‐throughput approaches for identification of target genes to resolve secretion bottlenecks in Chinese hamster ovary cells

Henning Gram Hansen1, Anne Mathilde Lund1, Håkan Jönsson1,2, Johan Rockberg1,2, Mikael Rørdam 1 1 Andersen and Helene Faustrup Kildegaard

1 The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark. 2 Royal Institute of Technology, Sweden

Chinese hamster ovary (CHO) cells are widely used in the biopharmaceutical industry as cell factories for the production of recombinant glycoprotein therapeutics. Before the mature therapeutic proteins are secreted from the CHO cell, the precursor polypeptides are subject to co‐translational translocation to the endoplasmic reticulum (ER) and post‐translational modifications and folding in the ER and Golgi. The secretion rate of therapeutic proteins is generally thought to be limited either by the translation or posttranslational machinery – also known as the secretion bottleneck. The aim of the present study is to identify target genes that are able to resolve secretion bottlenecks i.e. to increase the folding and secretion capacity of CHO cells. Combining split‐GFP complementation assay and microdroplet fluidics technology will enable us to sort high secretor cells in single‐cell droplets based on fluorescence intensity. Differentially expressed genes (target genes) in high secretor cells as compared to control cells will subsequently be identified using RNA‐Seq. The effect on secretion of the target genes will subsequently be analyzed in a high‐throughput overexpression secretion assay. This assay is currently being developed where we combine the split‐GFP technology, 96‐deepwell cultivation and 96‐well based cell counting. As a proof of concept study for the secretion assay, we will analyze the effect on secretion of published reference genes as well as genes differentially expressed in high producer monoclonal CHO cell lines. Overall, our target gene identification and verification platform holds the potential to unravel genes that can rebuild and enhance the secretory pathway machinery in CHO cells.

P9 Target specific integration in CHO cells mediated by CRISPR/Cas9 technology

Jae Seong Lee and Helene Faustrup Kildegaard

Technical University of Denmark, Denmark

Chinese hamster ovary (CHO) cells have been used as the predominant workhorses for production of recombinant therapeutic proteins with complex glycoforms. Traditionally, development of recombinant CHO cell lines rely on random integration of gene of interests (GOI) into the genome, causing unpredictable and heterogeneous expression levels. Subsequent high-throughput clone screening process is therefore necessary to select proper clones suitable for high and stable expression of proteins. As the draft genome information of CHO cells and targeted genome editing technologies are becoming available, rCHO cell line development based on site-specific integration have the potential to overcome the limitations of clonal heterogeneity. Here, we demonstrate efficient targeted gene integration into site-specific loci in CHO-S cell lines using CRISPR/Cas9 genome editing system and compatible donor plasmid harboring GOI and drug- resistance marker selection gene. Simultaneous introduction of active single guide RNAs, Cas9 nucleases, and donor templates have enabled insertion of a 3.5-kb gene expression cassette at defined loci in CHO-S cell lines, following a simple drug-selection. Our results can pave the way for the targeting of GOI to specific loci in CHO cells, increasing the likelihood of generating isogenic cell lines with consistent protein production.

Animal Cell Cultures - Expression and Engineering

P10 Accelerating Genome Editing in CHO Cells using CRISPR CAS9

Carlotta Ronda1,#, Lasse Ebdrup Pedersen1,#, Henning Gram Hansen1, Thomas Beuchert Kallehauge1, Michael J. Betenbaugh1,2, Alex Toftgaard Nielsen1 and Helene Faustrup Kildegaard1

1 The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Denmark.2 Johns Hopkins University, USA

# These authors contributed equally to the work

P11 Subcellular Location and Translational Efficiency of Recombinant mRNAs During Early Stage Recombinant Expression

Thomas Beuchert Kallehauge1, Mikael Rørdam Andersen1, Christian KrounDamgaard2, Helene Faustrup 1 Kildegaard

1 Technical University of Denmark, Denmark. 2 Aarhus University, Denmark

E‐mail: [email protected]

With the Chinese Hamster Ovary (CHO) cell line being the preferred mammalian production host for complex pharmaceuticals proteins, it presents itself as a candidate for extensive cellular engineering for improved API production and quality. The genetic modifications made are often directed towards improving the production of a specific product, which means that this specific strain may not be suited for expression of a different recombinant protein. In the work presented here, the objective was to improve the expression capacity of the CHO cell in a non product specific manner. This was done by addressing a central aspect of secreted recombinant proteins expression, namely the recruitment of the recombinant mRNA to the ER membrane. The study was directed towards the subcellular location of recombinant mRNA and its association with the translational apparatus.

The subcellular location of mRNA encoding recombinant EPO, Rituximab heavy chain, and Rituximab light chain was examined through cellular fractionation. The fractionation analyses showed that >90% of the EPO and Rituximab mRNA were located with the ER fraction, similar to endogenous control mRNAs directed towards the ER. Comparisons of the translational profile of the mRNA of EPO and a cytosolic reporter showed an equal association with polysomes. Western analyses, combined with proteasome inhibition, showed that no translation occurred into the cytosol. These data indicate efficient recruitment of recombinant mRNAs to the ER and translational stalling by the SRP pathway functions efficiently. All in all, the initial steps of the translocational pathway and the translational capacity of recombinant mRNAs seem to function as endogenous mRNAs in the examined cells.

Animal Cell Cultures - Expression and Engineering

P12 Engineering amino acid supply pathways in Chinese hamster ovary cells

Daniel Ley*, Helene F. Kildegaard, Mikael R. Andersen

Technical University of Denmark, Denmark. *E-mail: [email protected]

The Chinese hamster ovary (CHO) cell line is the predominant mammalian cell factory for production of therapeutic proteins. With the recent availability of the CHO-K1 genome sequence and CRISPR/Cas9 genome editing tools, the road has been paved for efficient cell line engineering of CHO cells.

In the present study, we aim to characterize the uptake and metabolism of amino acids in CHO cells and reengineer the metabolism to increase intracellular amino acid pools available for protein synthesis. For this, we reconstructed the amino acid supply pathways in CHO including transporters, anabolic and catabolic pathways, based on legacy knowledge from mouse. By comparative gene expression analysis of protein producing- and parental clones we identified gene candidates in amino acid catabolism for knockout.

Furthermore, we will identify possible limitations in amino acid uptake transport and tRNA expression by analysis of the gene expression landscape and metabolite profiling. In conclusion, the current study aims to investigate the potential for improved protein productivity in CHO cells through increased amino acid availability and provide novel physiological knowledge on amino acid metabolism.

P13 Recombinant protein expression using microbial expression systems

Søren M. Madsen, Astrid Vrang, Ole C. Hansen, and Mathias Fanø.

Bioneer A/S, Kogle Allé 2, Hørsholm, Denmark

Bioneer A/S is a Danish independent, research-based service company within biomedicine, biomedical technology and biotechnology. A prominent Bioneer service is early development technologies including gene cloning, production, purification and characterisation of recombinant proteins in a non-cGMP setup, using microbial and mammalian gene expression systems.

The P170 Expression System, developed by Bioneer, is a proprietary protein production technology based on the gram positive endotoxin-free bacterium Lactococcus lactis, which has proven useful for manufacturing of high value recombinant proteins. The P170 system is based on the lactate-inducible promoter P170 and allows secretion of the protein of interest to the culture medium simplifying the downstream process. The P170 system includes a variety of expression vectors as well as a number of optimized production strains, tailored to the P170 technology and fermentation procedures, using production media without animal derived components.

Bioneer has also an in-house developed IP-free E. coli (termed the “E-c system”) expression platform, which is based on the hybrid IPTG inducible tac promoter. The E-c system can be used in a GMP environment as all elements are well known and validated for GMP. It has been used for production of various gene products in yields exceeding grams pr. liter in batch fermentations. We perform scale-up of protein production to 15 L fermentation volume, and we develop and perform purification of recombinant proteins using state of the art technologies.

We also offer expertise in biochemical and biophysical characterisation of proteins and we perform method development, API characterisation and analysis for folding and degradation. Key applications are mainly for drug formulation development, drug stability assessment and drug comparability studies.

Animal Cell Cultures - Expression and Engineering

P14 Polyketide Synthases from Fusarium: Cloning, Expression and Purification for Structural Analysis

KASPER KVESEL1, REINHARD WIMMER1, JENS L. SØRENSEN1, FREDERIK T. HANSEN2, MICHAEL T. OVERGAARD1, HENRIETTE GIESE1, TEIS E. SONDERGAARD1

1Aalborg Univeristy, Denmar. 2Aarhus University, Denmark

Fungi produce a wide array of secondary metabolites, where some show bioactivity by help of a number of enzyme complexes. Polyketide synthases (PKS) are a class of multidomain enzymes, producing a class of secondary metabolites called polyketides1,2. Only few structures of PKS’s have been described, even fewer from fungi and none from Fusarium species. Multidomain proteins can be quite challenging to work with, which is why the project intends to solve the 3D-structures of single domains of PKS’s. In this project, we clone, express and purify the Acyl-carrier protein (ACP) domain from PKS6 in Fusarium graminearum for structural analysis through Nuclear Magnetic Resonance (NMR) and X-ray crystallography experiments.

The gene of the ACP domain of the PKS6 from F. graminearum was expressed in E. coli through a vector containing a His tag for purification and a TEV protease cleaving site. The expression was induced using Isopropyl β-D-1-thiogalactopyranoside (IPTG) and the ACP peptide purified as well as concentrated through steps of tag purification, dialysis, TEV-protease cleavage and spin column. Results were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) which showed that the ACP domain was successfully expressed, cleaved and almost purified. Since the purification was more or less a success, further structure analysis using nuclear magnetic resonance (NMR), circular dichroism (CD) and mass spectrometry (MS) was performed. NMR and CD indicates a possible α-helix structure which is expected2.

References

1) Hansen, F. T. et al., International Journal of Food Microbiology 155 (2012) 128-136

2) Keatinge-Clay, A. T., Nat. Prod. Rep. 29 (2012) 1050

P15 Fusarium graminearum PKS14 is involved in orsellinic acid and orcinol synthesis

Simon Hartung Jørgensen1, Rasmus John Norman Frandsen2, Kristian Fog Nielsen2, Erik Lysøe3, Teis Esben Sondergaard1, Reinhard Wimmer1, Henriette Giese1 and Jens Laurids Sørensen1

1Aalborg University, Denmark. 2Technical University of Denmark, Denmark. 3Bioforsk– Norwegian Institute of Agricultural and Environmental Research , Norway

The available genome sequences show that the number of secondary metabolite genes in filamentous fungi vastly exceeds the number of known products. This is also true for the global plant pathogenic fungus Fusarium graminearum, which contains 15 polyketide synthase (PKS) genes, of which only 6 have been linked to products. To remedy this, we focused on PKS14, which has only been shown to be expressed during plant infections or when cultivated on rice or corn meal (RM) based media. To enhance the production of the resulting product we introduced a constitutive promoter in front of PKS14 and cultivated two of the resulting mutants on RM medium. This led to the production of two compounds, which were only detected in the PKS14 overexpressing mutants and not in the wild type or PKS14 deletion mutants. The two compounds were identified as orsellinic acid and orcinol by comparing fragmentation patterns from high resolution mass spectrometry (HRMS) analyses and retention times to authentic standards. Orcinol was isolated from the PKS14 overexpressing mutant and the identity verified by NMR. Orcinol and orsellinic acid have previously primarily been detected in lichen fungi, but not in Fusarium. Orsellinic acid is hypothesized to be the PKS release product which is transformed to orcinol through decarboxylation. This is supported by phylogenetic analyses of PKSs, which placed PKS14 in a subclade of known OA synthases. Based on expression analyses the PKS14 gene cluster is predicted to include seven additional genes including a decarboxylase, which could be responsible for transforming orsellinic acid to orcinol.

Animal Cell Cultures - Expression and Engineering

P16 Expression, Purification and Characterisation of Domains from The Sarcoplasmic Calcium Release Channel RyR2

Christian Holt, Louise Hamborg Nielsen, Kamilla Taunsig Larsen and Heidi Øllegaard Johnsen

Aalborg University, Denmark

Cardiac contraction is a result of calcium release from internal calcium stores – the SR. This release is governed by large (>2 MDa) homotetrameric calcium release channels named Ryanodine Receptors (RyRs). Mutations in these channels, and in the calcium sensor protein calmodulin (CaM), have been linked to catecholaminergic polymorphic ventricular tachycardia (CPVT). CPVT is a genetic cardiac disorder, characterised by adrenergic induced ventricular fibrillations that can lead to syncope and in some cases sudden cardiac arrest.

The regulation of RyR activity and its mechanism of action in calcium release is not fully understood. In order to gain further insight into this, a small part of the calcium release channel was investigated by expressing and characterising selected domains in RyR2.

Following ligation independent cloning using pMAL vectors, a putative calcium binding EF hand motif from the cardiac RyR2 isoform was expressed independently and coherent with the adjacent RyR and IP3R homology (RIH) domain. A purification procedure was established before the stability and protein-ligand interaction of the pure recombinant proteins are analysed. The unfolding transitions of the purified protein domains were examined by thermal denaturation using fluorescence, while circular dichroism was used to determine the secondary structure content. Furthermore, a ligand interaction study was performed by anisotropy measurements using assays with calcium and EDTA, respectively, and a RyR2 CaM binding domain (CaMBD2) labelled with the TAMRA fluorophore.

We demonstrate the presence of α-helical structure, indicating that the purified RIH-EF domain is folded. Thermal denaturation revealed a melting temperature of 37 oC supporting the presence of secondary structure, with a marginal stability. Furthermore, a protein-ligand interaction of both fusion proteins with CaMBD2 was demonstrated, both in the presence and absence of calcium, suggesting that these domains may be important for the mechanism of CaM regulation of RyR activity.

P17 CRISPy : A TOOL FOR CRISPR CAS9 GUIDE RNA SELECTION IN CHO

Lasse Ebdrup Pedersen1#, Carlotta Ronda1,#, Henning Gram Hansen1, Thomas Beuchert Kallehauge1,

Michael J. Betenbaugh1,2, Alex Toftgaard Nielsen1 and Helene Faustrup Kildegaard1

1Technical University of Denmark, Denmark, 2Johns Hopkins University, USA

#These authors contributed equally to the work

Background: We wished to test and, if possible, use the crispr cas9 system for genome engineering in CHO cells. The potential of the Cas9 system to dramatically reduce cost and simultaneously increase efficacy makes this a highly desirable goal. A key requirement in using this system is identification of potential Cas9 gRNA target sites and an evaluation of potential offtarget sites.

Experimental design: To meet these requirements I created a database of potential cas9 gRNA binding sites using the CHO-K1 genome. Each site was also evaluated according to their uniqueness compared to the rest of the CHO-K1 genome . A high degree of uniqueness decreases the likelihood of undesired genome modifications at unintended locations. To browse this database I created a web based interface, CRISPy, that allows the user to quickly identify potential sites in a gene of choice.

Results and Discussion: We have successfully used CRISPy to design 96 gRNA's (8 targets in each of 12 genes) and PCR primers to test each gRNA. This took ~15min per gene. New features are being added as new research demonstrates improved design schemes. Currently CRISPy features:  Gene intron-exon overview and exon by exon detailed view  Sorting according to fewest potential offtargets  Shopping cart system for evaluating multiple target sites  Can identify potential double nicking partners to further decrease offtarget effects  Pre-configured links to NCBI primerblast to facilitate easy design of PCR that can be used to test genome editing at desired site as well as offtarget sites.

Animal Cell Cultures - Expression and Engineering

Exhibitors

Animal Cell Cultures - Expression and Engineering

Thermo Fisher Scientific

Att.: Willy Bjørklund, Stamholmen 193, 2650 Hvidovre, Denmark

Phone: +45 2227 1991 e-mail: [email protected]

Svanholm.com

Att.: Bent Svanholm, 4760 Vordingborg, Denmark

Mobile: +45 7026 5811 e-mail: [email protected]

Nordic BioSite ApS

Att.: Ulla Lauridsen, Toldbodgade 18,5., 1254 Copenhagen K, Denmark

Phone: +45 2484 8485 e-mail: [email protected]

Dandiag A/S

Att.: Kirsten Thuesen, Mårkærvej 9, 2630 Tåstrup, Denmark

Phone: +45 2091 4000 e-mail: [email protected]

A/S Ninolab

Jesper Secher, Værkstedsvej 24C, 4600 Køge, Denmark

Mobile: +45 4491 1007 e-mail: [email protected]

Animal Cell Cultures - Expression and Engineering

Insatech A/S

Att.: Kenneth Rasmussen, Algade 133, 4760 Vordingborg, Denmark

Phone: +45 2085 6068 e-mail: [email protected]

In Vitro

Att.: Pia Brevadt & Fadi Muhiddine, Kratbjerg 336, 3480 Fredensborg, Denmark

Phone: +45 4847 5070 e-mail: (Kirsa Schalck) [email protected]

Holm & Halby

Att.: Mette Bursell, Vallensbækvej 35, 2605 Brøndby, Denmark

Phone: +45 4326 9400 e-mail: [email protected]

Bio-Rad Laboratories, Life Sciences

Att.: Gunnar Warnke, Symbion, Fruebjergvej 3, 2100 København Ø, Denmark

Phone: +45 3033 9402 e-mail: [email protected]

List of Participants

Animal Cell Cultures - Expression and Engineering

Name Institution/ Department E-mail Company

Ali Kazemi Seresht Novo Nordisk A/S Cell Culture [email protected] Technology

Alison Lilley University of Novo Nordisk [email protected] Copenhagen Foundation Centre for Protein Research

Anders Vagnø ALK A/S Pharmaceutical [email protected] Pedersen development, R&D

Anders Aamann Roche Diagnostics [email protected] Rasmussen A/S

Anja Mathiesen Molecular Devices [email protected]

Anne Mathilde Technical Systems Biology [email protected] University of Denmark

Anne Pihl Bali Technological Systems Biology [email protected] University of Denmark

Bent Svanholm Svanholm.com / [email protected] Technical University of Denmark

Bjørn Voldborg Technical NNF Center for [email protected] University of Biosustainability Denmark

Carola Heesemann Technical [email protected] University of Denmark

Carsten Hjort Novozymes A/S Production Strain [email protected] Technology

Christian Brøchner Danish Stem Cell [email protected] Society (DASCS)

Christian Biogen Idec Manufacturing [email protected] Christensen Sciences

Christian Clausen Danish Stem Cell [email protected] Society (DASCS)

Christian Holt Aalborg University Biotechnology, [email protected] Chemistry and Environmetal Engineering

Christian Müller CMC Biologics [email protected]

Christina Rottbøll Symphogen Cell Line Generation [email protected] Andersen

Christoffer Bro Biogen Idec Manufacturing [email protected] Sciences

Christophe Madsen Danish Stem Cell [email protected] Society (DASCS)

Animal Cell Cultures - Expression and Engineering

Name Institution/ Department E-mail Company

Daniel Ley Technical Systems biology [email protected] University of Denmark

Dennis Kristiansen Aalborg University Biotechnology, [email protected] Chemistry and Environmetal Engineering

Eleftherios (Terry) University of Chemical & [email protected] Papoutsakis Delaware Biomolecular Engineering

Emil Poulsen Biogen Idec Manufacturing [email protected] Sciences

Eric Paul Bennett University of Center for [email protected] Copenhagen Glycomics, Department of Odontology

Fadi Muhiddine In Vitro [email protected]

Fredrik Kartberg H. Lundbeck A/S Biologics [email protected]

Giuseppe Cazzamali faculty of CPR (center for [email protected] medcine, protein research) Copenhagen universtiy

Gunnar Warnke Bio-Rad [email protected] Laboratories, Life Sciences

Gyun Min Lee KAIST Biological Sciences [email protected]

Hans Erik Mølager Technical [email protected] Christensen University of Denmark

Hans Peter Taconic Europe [email protected] Sørensen A/S

Heidi Øllegaard Aalborg University Biotechnology, [email protected] Johnsen Chemistry and Environmetal Engineering

Helene Faustrup Technical Novo Nordisk [email protected] Kildegaard University of Foundation Center Denmark for Biosustainability

Henning Gram Center for CHO Cell Line [email protected] Hansen Biosustainability, Engineering and Technical Design University of Denmark

Name Institution/ Department E-mail Company

Holti Kellezi NNF Basic Integrative [email protected] Metabolic Physiology Research (University of Copenhagen)

Inge Kjaerboelling Technical Systems Biology [email protected] University of Denmark

Jae Seong Lee Technical DTU Biosustain [email protected] University of Denmark

Jakob Olsen Biogen Idec Manufacturing [email protected] Sciences

Jens Christian Technical Systems Biology [email protected] Eriksen University of Denmark

Jens Gram CMC Biologics A/S Process Transfer [email protected]

Jesper Secher A/S Ninolab [email protected]

Jochen Förster Technical Applied Metabolic [email protected] University of Engineering Denmark / Novo Nordisk Foundation Center for Biosustainability

Johnny Arnsdorf The Novo Nordisk CHO CORE [email protected] Foundation Center for Biosustainability

Jonathan D. Thermo Fisher Life Sciences [email protected] Chesnut Scientific Solutions Group, Synthetic Biology R&D

Kai Touw Crucell (a Johnson Vaccine Process & [email protected] & Johnson Analytical Company) Development

Kamilla Taunsig Aalborg University Biotechnology, [email protected] Larsen Chemistry and Environmetal Engineering

Kasper Kvesel Aalborg University Biotechnology, [email protected] Chemistry and Environmental Engineering

Kenneth Rasmussen Insatech A/S [email protected]

Kevin Kayser Sigma-Aldrich [email protected]

Kirsten Thuesen Dandiag A/S [email protected]

Animal Cell Cultures - Expression and Engineering

Name Institution/ Department E-mail Company

Krzysztof University of TCEL-group, [email protected] Wrzesinski Southern Department of Denmark - SDU Biochemistry and Molecular Biology

Lars Haastrup Aalborg University Biotechnology, [email protected] Pedersen Chemistry and Environmetal Engineering

Lasse Ebdrup Technical NNF Center for [email protected] Pedersen University of Biosustainability Denmark

Lea M. Madsen Technical Systems Biology, [email protected] University of NNF Center for Denmark Biosustainability

Leif Schauser CLC bio [email protected]

Linda Rojek Jensen Technical Systems Biology [email protected] University of Denmark

Louise Hamborg Aalborg University Biotechnology, [email protected] Chemistry and Environmetal Engineering

Malene Brohus Aalborg University Biotechnology, [email protected] Chemistry and Environmetal Engineering

Malene Tving Thermo Fisher [email protected] Jensen Scientific

Marianne Decker The Novo Nordisk CHO CORE [email protected] Foundation Center For Biosustainability

Marianne S. Danish Stem Cell [email protected] Andersen Society (DASCS)

Marlene Petersen Dandiag A/S [email protected]

Martin Bonde DANSK BIOTEK [email protected]

Martin Dufva Technical DTU Nanotech [email protected] University of Denmark

Mette Rahbek Thermo Fisher Biosciences [email protected] Christoffersen Scientific

Michael Johns Hopkins Department of [email protected] Betenbaugh University Chemical and Biomolecular Engineering

Michael Wahlers Biogen Idec Manufacturing [email protected] Sciences

Name Institution/ Department E-mail Company

Michael Williamson Copenhagen NNF Center for [email protected] University Protein Research

Mikael Rørdam Technical Department of [email protected] Andersen University of Systems Biology Denmark

Mikkel A. Danish Stem Cell [email protected] Rasmussen Society (DASCS)

Nicole Borth BOKU University Department of [email protected] Vienna Biotechnology

Niels Skjærbæk Bioneer A/S [email protected]

Nikolaj Rasmussen Biogen Idec Manufacturing [email protected] Sciences

Nils Færgeman University of Biochemistry and [email protected] Southern Molecular Biology Denmark

Ole Jakobsen Bio-Rad [email protected] Laboratories

Per Juul Holm & Halby [email protected]

Per Stobbe CerCell [email protected]

Peter Ravn Bioneer A/S [email protected]

Pia Brevadt In Vitro [email protected]

Poul Baad CMC Biologics A/S Upstream [email protected] Rasmussen Development

Preben Kjemtrup Pall Norden Sales [email protected]

Rob Pickles Roche Diagnostics [email protected] A/S

Robert Oscar Svanholm.com [email protected] Burdorf

Sara Petersen Bjørn The Novo Nordisk CHO Core Cellline [email protected] Foundation Engineering Center for Biosustainability

Simon Hartung Aalborg University Biotechnology, [email protected] Jørgensen Chemistry and Environmental Engineering

Stefan Kol The Novo Nordisk CHO cell line [email protected] Foundation engineering core Center for Biosustainability

Stephanie Mesker Technical [email protected] University of Denmark

Stephen John Fey University of TCEL-group, [email protected] Southern Department of Denmark - SDU Biochemistry and Molecular Biology

Animal Cell Cultures - Expression and Engineering

Name Institution/ Department E-mail Company

Svend Kjær Cancer Research Protein Purification [email protected] UK Facility

Søren Madsen Bioneer A/S Bacterial Expression [email protected]

Thomas Kallehauge Novo Nordisk CHO Cell Line [email protected] Foundation Engineering Centre for Biosustainability

Thomas Schou Science & Biopeople [email protected] Larsen Innovation Innovation Network Network Manager

Tom Kristensen Biogen Idec Manufacturing [email protected] Sciences Ulla Laursen Nordic Biosite [email protected]

Vasco Botelho University of TCEL-group, [email protected] Carvalho Southern Department of Denmark - SDU Biochemistry and Molecular Biology

Wagma Saei Aalborg University Biotechnology, [email protected] Chemistry and Environmetal Engineering

Wian de Jongh ExpreS2ion [email protected] Biotechnologies

Willy Björklund Thermo Fisher [email protected] Sceintific

Yuzhou Fan Technical [email protected] University of Denmark and Symphogen A/S

Yves Durocher National Research Human Health [email protected] Council Canada Therapeutic Portfolio - Biologics & SEBs Program

Zhang Yang University of Copenhagen Center [email protected] Copenhagen for Glycomics, ICMM