CCDWC 2017 Day 2

Upstream – CRISPR / Cell line development

Upstream - Media and process development

Downstream – Advances in chromatography and novel purification methods



From thousands of clones to the final one

  • CHO cells are the most widely used host for large-scale production of recombinant therapeutic proteins
  • In order to identify the most suitable lead cell lines Novartis started to introduce a development process that comprises thorough early candidate profiling focusing on biophysical properties of candidates, followed by a cell line development process
  • applying an efficient selection strategy in combination with process-tailored automation for clone imaging, clone handling and selection

Cell line development and process development for biosimilars

  • Definition of Development goals
  • Choice of cell lines
  • Quality Attributes during Process Development

Continuous chromatography – challenging bioprocess development problems

  • Continuous operation of polishing chromatography
  • Robustness and fault diagnosis of continuous chromatography
  • Integration of continuous steps

Lyophilization in Dual Chamber Cartridges – from Energy Transfer to Process Control

  • Process challenges during freeze-drying in DCCs
  • Selection of adequate holder devices
  • Process design and control with the “DCC-LyoMate procedure”

CHO cell factory engineering, for working towards improved production of therapeutic proteins

  • CHO cell line engineering and therapeutic protein production
  • CRISPR/Cas9-mediated genome engineering technologies
  • Targeted integration to facilitate controlled and reproducible cell line engineering

Increase yields via media optimisation toolbox and process parameters improvement

  • A custom media optimization approach was designed for use with the ambr® 15 microbioreactor system
  • Fed-batch processes were developed for multiple case studies for cell lines producing a variety of antibodies
  • Media optimizations and improvements in peak viable cell density and titer will be compared between culture systems and case studies

Challenging biomolecule purifications resolved with innovative selectivities

  • Novel antibody formats like fragments or non-IgG molecules, lack a strong protein A binding site facilitating efficient capture and purification in a protein A affinity based platform.
  • New developments in ligand technology provi10:00de chromatographic materials with unique selectivity as well as high capacities (ligand grafting)
  • Discussion will demonstrate the development of a purification platform for non-IgG type antigen binding biopharmaceuticals using these innovative selectivities

Mabs aggregation due to mechanical stress during fill & finish process

  • Mixing: classical bottom agitation may induce very high stress for particular fragile Mabs, and this is the reason why technology of agitation by levitation is spreading in Biotech processes . Very simple lab-scale studies can demonstrate how even low contact between a fix surface and a mobile agitator generate high amount of particulates.
  • Filling: in a same manner, piston pump filling implies contact and movement between two stainless steel parts and generates significant quantity of particulates, comparatively to less stressful technologies like peristaltic pump or time-pressure.
  • The potential root cause of these Mab degradations occurring during mixing with contact and piston pump filling will be discussed: shear stress, abrasion and formation of material particulates, etc

Rapid cell line development and efficient genome modification using scalable electroporation

  • Flow electroporation transfection technology enables highly efficient loading of plasmids, messenger RNA and other molecules into cell lines and primary cells at scales ranging from <1e6 to 2e11 cells
  • Discuss how the high efficiency and viability allows generation of stable pools and clones with reduced time and effort
  • Share data on efficient targeted modification of mammalian genomes for cell therapy and protein expression using CRISPR/Cas-9 and zinc finger nucleases

Characterisation of the phosphoproteome of recombinant Chinese hamster ovary (CHO) cells to improve bioprocess-relevant phenotypes

  • Phosphorylation is a key post-translational modification, playing a crucial role in regulating many cellular processes including transcription, translation, signaling, etc
  • The impact of the phosphoproteome on bioprocess performance is poorly characterized in recombinant CHO cells
  • We have identified over 700 differentially expressed phosphopeptides using quantitative label-free LC-MS/MS phosphoproteomic analysis in conjunction with IMAC and TiO2 phosphopeptide enrichment strategies, following a reduction in culture temperature (temperature shift)
  • Phosphoproteins have the potential to be cell engineering targets to improve bioprocess-relevant phenotypes

3D printing of chromatographic columns

  • 3D printing as a tool to revolutionize the design chromatographic columns
  • Closing the loop between experiments and models
  • Direct capture of proteins contained in solid-laden streams

Lyophiliser characterisation: determination of equipment heat and mass transfer properties to support scale-up and technical transfer of lyophilised biopharmaceuticals

  • Lab and commercial scale determination of vial heat transfer coefficient and sublimation behaviour
  • Predicting primary drying conditions in different lyophilisers
  • Design space generation
  • Utilising knowledge of equipment and product to support manufacturing

10:00 Networking Break


Toolbox for Cell Line Development – Next-Generation Cell Line Development Technologies

  • CHO cells are the most widely used host for large-scale production of recombinant therapeutic proteins.
  • A novel toolbox of vector elements, selection marker and novel engineered CHO cell lines were developed which results in combination in significant increase of titer and improved product quality.
  • We have identified a key protein severely affecting the quality of non-antibody format therapeutic proteins.
  • Elimination of this protein via novel targeted gene disruption tools resulted in significant increased improved product quality.

Recent innovations in liquid media preparation

  • Making liquid large volume media preparation more efficient
  • Recent innovations in liquid media preparation
  • De-bottlenecking large volume liquid media preparation

Excipient considerations in lyophilised protein formulations

  • Commercial protein formulations: a review of excipients
  • Impact of arginine on the freeze drying performance of protein formulations

Downstream - PAT, process modelling, monitoring and control


Beyond GS: Using different genome editing techniques to generate improved commercially available production cell lines

  • Genome engineering has significant potential to improve our ability to manufacture biotherapeutic products
  • CRISPR technology can be applied to perform rapid proof of concept edits
  • After validation, Horizon uses rAAV technology to generate improved CHO cells for commercial release with a clear IP position
  • Here we present data on edits that build on our GS null cells to create our next generation cell line platform

Proteomic identification of bioprocess markers for cell stress and cell death during perfusion culture

  • Lab-scale model system for apoptotic and necrotic CHO
  • Screening and targeted LC-MS workflow for the identification of protein viability markers from apoptotic and necrotic CHO
  • Method application to samples from biomanufacturing

Applications of Process Analytics in Continuous Downstream Processing

  • Conti flow manufacturing can have an enormous impact on manufacturing in terms of quality, quality and cost
  • Moreover it is possible to design conti flow systems that are inherently more controllable than batch processes
  • To take advantage of these control possibilities requires us to develop robust and reliable real-time process measurements

End of Formulation, Fill & Finish Track


miRNA engineering of CHO host cell lines facilitates production of difficult-to-express proteins to increase success in cell line development

  • MicroRNAs (miRNAs) have emerged as potent cell engineering tools to improve bioprocess performance of CHO cells. We established a novel engineered CHO host cell line constitutively expressing a pro-productive miRNA in order to enhance overall bioprocess performance.
  • Novel miRNA engineered host cells were tested in two independent CLD campaigns using two different mAb candidates including a difficult-to-express antibody. Production clones derived from target miRNA overexpressing CHO host cells outperformed non-engineered control cells and exhibited substantially increased product yields without compromising product quality. 
  • Our results demonstrate that host cell engineering using miRNAs represents a promising tool to overcome limitations in industrial bioprocess development especially regarding challenging proteins.

Discussion: Media and process discussion and extended Q&A

  • Discussion to talk about media and process development across the cell culture industry
  • Key challenges that companies and academics are facing
  • New movements in this area
  • Extended Q&A to the speakers

A novel toolbox for monitoring inclusion body processing

  • Novel toolbox comprising HPLC and chemometric tools to monitor the complex steps in a typical inclusion body process
  • Toolbox allows the analysis of these steps within minutes after sampling, thus allowing decisions when a unit operation is done
  • Reduces overall process time and costs, and also minimises the risk of product loss

Upstream - Innovation showcase


Benefits of a platform technology approach for the generation of production cell lines and processes

  • Description of CHO based platform technology including essential key components
  • Description of Cell line development process and selection strategy to select for high

The C1 expression system – A journey to displace CHO cells with breakthrough cell expression

  • Producer of enzymes and proteins using a proprietary and patented expression system based on the Myceliopthora thermophila fungus, nicknamed C1.
  • A hyper-productive fungal expression system used to develop & manufacture large quantities of desired proteins at industrial scale at significantly lower CapEx and OpEx costs.
  • Can produce Vaccines at high level and with excellent immune response - Dyadic has also displayed the ability to easily express mAb’s.

Getting the full picture: Addressing Protein Stability by nanoDSF

  • How to make better informed decisions earlier in the biologics development workflow
  • Explore the modern alternative to DSC: 100x faster, 40x less sample
  • Predicting stabilities: thermal and chemical unfolding, melting points and deltaG, aggregation behaviour

12:05 Networking Lunch


Ultra-deep next generation mitochondrial genome sequencing reveals widespread heteroplasmy in Chinese hamster ovary cells

  • First mitochondrial genome sequence of the Chinese hamster
  • Widespread heteroplasmy across a panel of Chinese hamster ovary cell lines
  • At least one heteroplasmic variation/cell line which resulted in an amino acid change
  • Potential impact on CHO cell metabolism

Use of PAT for process development and control – Toward process digitalization

  • Overview on statistical tools for process screening, development, monitoring and control. Where can we save money and how to use such tools for quality management?
  • Use of online monitoring and supervisory control for process development. Is it too early?
  • Introduction to advanced methods for process development: hybrid models. How to effectively manage knowledge during process development?
  • Future perspectives on process digitalization
Michael Sokolov, PhD Candidate, ETH Zurich

microRNAs for optimized production: towards a deeper understanding of underlying mechanisms

  • miRNAs represent promising tools for cell engineering of biopharmaceutical production cells
  • Easy-to-use methodology makes miRNAs feasible for usage in production process
  • Detailed analyses of underlying mechanisms of action within cells can reveal pathways mediating improved phenotypes

A holistic approach to analytical support, for the biotech industry

  • Growing demand for new analytical tools;
  • Increasing infrastructure costs;
  • Subcontracting of analytical work

Upstream – Continuous manufacture

Dr Berthold Boedeker

Update on ballroom concept facilities for continuous manufacture

  • Status and limitations of continuous processing
  • Status implementation of disposables and closed processing concept
  • Ballroom plant design for continuous processing
Martin Kornecki

USP/DSP integration

  • Upstream strategies that shift the focus from high titer only to titer in respect to the processability in the downstream
  • Increasing titer lead to even more increasing side components, making the downstream very complex and therefor the whole process more expensive than a medium titer with good to separate side components
  • Biothermodynamics and online control strategies for the fermenter

14:20 Networking Break

Closing plenary: Integrated processing

Professor Christoph Herwig

Development and characterization of integrated processes using data science tools

  • Identification of interaction effects between unit operations
  • Similarity analysis of integrated processes for technology transfer
  • Root cause analysis for bioprocesses
  • Solutions for process characterization and process validation along the new validation guideline Annex 1 and  CPV
Dr Alessandro Butte

A financial perspective to integrated bioprocessing – Can we make this cost effective?

  • Overview on integrated continuous bioprocessing, from an operational and financial perspective
  • Cost of good analysis and comparison to standard batch processes. Where do we stay and where should we focus
  • Investment (net present value) analysis on integrated process for multi-drug production plants: do we really lose in flexibility?
  • Future perspectives: How can industry work together a holistically integrated process? What is still needed to exploit this technology?

16:30 End of Conference

last published: 17/Feb/17 11:15 GMT