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Proteins for structural and biological studies

15th Annual Protein Expression Workshop

30 July – 1 august 2014https://wiki.csiro.au/display/PEWs/PEWs+Home

www.csiro.au

Major sponsors

15th Annual Protein Expression WorkshopProteins for structural and biological studies

IntroductionThe CSIRO Protein Expression Workshop is an annual event designed to improve the understanding of proteins for structural and biological studies. Join us for the 15th Annual Protein Expression Workshop from 30 July – 1 August 2014.

Workshop aimsThe production of complex proteins for structural and biological studies requires a range of skills and disciplines including molecular biology, fermentation, protein purification and structural analysis. This workshop will cover a range of topics to enable successful production work and provides an excellent networking opportunity.

www.csiro.au

https://wiki.csiro.au/display/PEWS/PEWS+Home

By the end of the Workshop participants will:• have an understanding of different expression

systems

• be familiar with basic technologies involved in protein production

• know the most important large and small-scale equipments involved in expression and purification

• be able to design and optimise your production system

• understand basic purification and assay methods

• learn how to integrate your production system with purification

• be familiar with current trends in protein production and fermentation

• learn how collaborative facilities might be able to assist you to reach your research goals.

Gold sponsors

Silver sponsors

Major sponsors

b Blueprints for a sustainable future

Bronze sponsors

Hands-on Workshop sponsored by

www.biosci.com.au

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PROTEINS FOR STRUCTURAL AND BIOLOGICAL STUDIES

15th Protein Expression Workshop • https://wiki.csiro.au/display/PEWS/PEWS+Home

DAY ONE : Wednesday 30th July 2014

Venue CSIRO - 343 Royal Parade, Parkville

8:30AM Registration

9:15AM Safety induction and housekeeping

SETTING THE SCENE Chair: Greg Coia

9:20AM Introduction and welcome Paul Savage (Manufacturing Flagship)

9:30AM OPT-302: A novel VEGFR-3 fusion protein for the treatment of wet AMD

Mike Gerometta (Circadian Technologies Ltd)

10:00AM Non-native and modified particulate immunogens with enhanced immunogenicity

Hans Netter (Monash University)

10:30AM MORNING TEA

11:00AM Helicobacter: Then and Now Barry Marshall (University of Western Australia)

12:00PM Rotavirus vaccine development Rajendra Sabale (Serum Institute of India)

12:30PM LUNCH

PROTEIN EXPRESSION Chair: Ian Macreadie

1:30PM Systems for large-scale manufacture of vaccines and biopharmaceuticals

John Power (Zoetis)

2:00PM Strategies for expressing recombinant proteins in bacteria and their purification

Bill McKinstry (CSIRO)

2:30PM Improving Secretion and Purification of Glycoproteins in the Baculovirus-Insect Cell Expression System

Cindy Chang (UQ-PEF)

3:00PM Yeast expression systems Brendon Monahan (CSIRO)

3:30PM AFTERNOON TEA

4:00PM Mammalian toolbox – Platform technology for developing human therapeutics

Jeff Hou (AIBN)

4:30PM Discovery and development of novel human therapeutics using recombinant protein expression in mammalian cells

Catherine Owczarek (CSL)

5:00PM CareerLounge: Connecting students, employers and educators

Dominique Fisher (CareerLounge)

5:30PM Discussion and close of Day 1

DRINKS AND PIZZAS Sponsored by BPN

Program

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Program


DAY TWO : Thursday 31st July 2014

Venue CSIRO - 343 Royal Parade, Parkville 9:00AM Registration

9:25AM Welcome Tim Adams (CSIRO)

9:30AM Control of Glycosylation Profiles in Cell Bioprocesses Mike Butler (Univ of Manitoba) PROTEIN PURIFICATION & CHARACTERISATION Chair: Lou Fabri

10:00AM Purification and characterisation of mammalian expressed proteins: key assessment considerations for candidate/target selection in a research based commercial environment

Pierre Scotney and Peter Schmidt (CSL)

10:30AM MORNING TEA

11:00AM Structural Biology at the Australian Synchrotron Jose Varghese (CSIRO)

11:30AM Simple and low cost quality control of expressed proteins by mass spectrometry

Nick Williamson (Bio21/Melbourne University)

12:00PM Physical techniques in a protein workflow Shane Seabrook (CSIRO)

12:30PM LUNCH

COLLABORATIVE CENTRES & SUCCESS STORIES Chair: Jan Tennent

2:00PM Australia Therapeutics Pipeline Stewart Hay (TIA)

2:15PM The National Biologics Facilities (NBF) – A joint AIBN/CSIRO Facility

Peter Gray (AIBN/UQ)

2:30PM BPA and the Biologics Facility of the Future Brett Whitecross (BPA)

2:45PM The Victorian Antibody Initiative (VAI) Caroline Laverty (MATF)

3:00PM GMP Considerations for recombinant protein manufacture and working with a CMO

Brett Butcher (PharmaSynth)


4:00PM Insulin receptor: production of midi-, mini- and micro-constructs: Targeted design of insulin receptor for crystallography of Site 1

John Menting (WEHI)

4:30PM Telomerase: a promising target for cancer Scott Cohen (CMRI)

5:00PM Discussion and close of Day 2

5:30PM PRE-DINNER DRINKS

Sponsored by BioMedVic

7:00PM CONFERENCE DINNER Fish Bowl at the Melbourne Aquarium

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Program


DAY THREE : Friday 1st August 2014

Venues CSIRO Ian Wark Lab Reception Bayview Avenue, Clayton

CSIRO Parkville Laboratories 343 Royal Parade, Parkville

8:40AM Meeting at Parkville reception for bus ride to Clayton workshops

n/a

9:00AM n/a Welcome to Parkville & Safety Induction (2nd floor Seminar room)

9:15AM Welcome to Clayton & Safety Induction (Ian Wark Canteen)

9:30PM WORKSHOP STARTS

10:40AM MORNING TEA

11:00PM WORKSHOP CONTINUES 12::30PM LUNCH

1:30PM WORKSHOP CONTINUES


3:15PM WORKSHOP CONTINUES 4:00PM Bus leaves to go back to Parkville n/a 4:30PM CLOSE OF WORKSHOP CLOSE OF WORKSHOP

WORKSHOP 1: Microbial Scale-up Bioreactor assembly, probe calibration, sampling and process monitoring. Including demonstration of the Sartorius BioStat B benchtop bioreactor. Geoff Dumsday Linda Howell Andreas Kocourek Kanika Bhadoria

WORKSHOP 3: Protein Purification and Characterisation Participants will have the chance to attend 2 of the following 6 workshops: Mini Workshop 1: Akta-Xpress (GE) (3h) Lesley Pearce/David Cossens Mini Workshop 2: Profinia/Duo-Flow (BioRad) (3h) John Bentley/Nishen Naidoo Mini Workshop 3: His-tagged protein purification (3h) Pat Pilling Mini Workshop 4: Mass Spectrometry (1.5h) Lindsay Sparrow/Nick Bartone Mini Workshop 5: Size-Exclusion Chromatography (ATA) (3h) Paul Barrett Mini Workshop 6: BLItz Sytems (Millenium Science) (3h) Mike Yarski

WORKSHOP 2: Mammalian Cell Culture Mylinh La and Louis Lu ReadyToProcess WAVE™ 25 (GE) John Ince Cytell Cell Imaging System (GE) Tracee Archibald

Chemidoc for V3 Western Workflow (BioRad) Bansi Sanghvi

Transient transfection/Cell count and analysis (Life Technologies)

Alison Digney and Naomi Morisson Single-use Bioreactor system (Eppendorf) Stephen de Lacey

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Notes

DAy onE Presenters

INtroductIoN aNd Welcome

PAUL SAVAGECSIRO Manufacturing Flagship

Dr Paul Savage is the research director for the Biomanufacturing Program in the Manufacturing Flagship at CSIRO. His program provides scientific capabilities in support of companies involved in biotechnology and chemical innovation. This includes the design, construction and process tech transfer for biological and synthetic molecules of potential commercial importance.

The program primarily uses the science capabilities of:

• molecular and cell biology including enzymology

• stem cell biology and scale-up

• bacterial and mammalian cell fermentation up 500L scale and downstream processing

• protein science including structural biology and X-ray crystallography

• synthetic organic chemistry including scale up and process chemistry

• flow chemistry and high-throughput synthesis

Paul is working at the interface between business and science to help build a vibrant, globally successful Australian biotech industry.

oPt-302: a novel VeGFr-3 fusion protein for the treatment of wet amd

MikE GEroMEttA Circadian Technologies Ltd

OPT-302 is a soluble form of the vascular endothelial growth factor receptor-3 (VEGFR-3) expressed as an Fc-fusion protein. OPT-302 functions by capturing and thereby neutralizing the activity of VEGF-C and VEGF-D on the endogenous receptors VEGFR-2 and VEGFR-3. OPT-302 is highly specific for VEGF-C and VEGF-D, and does not bind VEGF-A. Administration of OPT-302 can inhibit blood and lymphatic vessel growth, as well as vessel leakage, which are characteristic hallmarks of several eye diseases, including neovascular (“wet”) macular degeneration (AMD).

The molecular targets for OPT-302, VEGF-C and VEGF-D, differ from that of marketed biologic VEGF-A inhibitors, such as ranibizumab and aflibercept, which bind VEGF-A and prevent the interaction of VEGF-A with its receptors (VEGFR-1 and VEGFR-2) on the surface of endothelial cells, reducing endothelial cell proliferation, vascular leakage, and new blood vessel formation. Therefore, the therapeutic activity of OPT-302 may be complementary to that of existing therapies for eye disease.

OPT-302 has been designed as an optimal ophthalmic agent for intravitreal administration. It has high affinity for its cognate ligands, a long ocular half-life potentially enabling a reduced administration regime, rapid blood clearance to minimize systemic adverse events and employs endogeneous proteins for reduced immunogenicity. The molecular structure also facilitates protein expression and purification. These properties have been displayed in pre-clinical models which show that OPT-302 inhibits wet AMD either alone or in combination with existing drugs.

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15th AnnuAl Protein exPression WorkshoP

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Biography

Dr Mike Gerometta has been Head of Chemistry, Manufacturing & Controls (CMC), Development since 2008 with responsibilities encompassing the outsourcing of Circadian’s research and cGMP manufacturing activities for its therapeutic and diagnostic portfolio, as well as oversight of internal laboratory activities. Mike over 25 years’ experience in the Australian biotechnology industry, most recently as Chief Operating Officer of Q-Gen, the manufacturing facility of the Queensland Institute of Medical Research. He has extensive experience working with numerous Contract Manufacturing Organisations overseas and locally in all facets of translational CMC from concept through to Phase II studies, in the process successfully guiding the manufacture of four biologics through to Phase 1/2 clinical trials, including oversight of two non-clinical programs, as well as associated regulatory interactions in North America and Australia. He has also directed the development of numerous in vitro diagnostic products through to the market over 19 years at Agen Biomedical, ultimately as Research and Product Development Director.

Mike was awarded his PhD in biotechnology from the Queensland University of Technology and has a degree in chemistry from the University of Technology in Sydney.

Non-native and modified particulate immunogens with targeting capability and enhanced immunogenicity

HAnS J. nEttEr Monash University

Virus-like particles (VLPs) are tools of a leading innovative bio-nanotechnology in vector and vaccine development. VLPs composed of the small envelope protein (HBsAgS) from hepatitis B virus represent a class of bio-nanoparticles, which are formed by viral structural proteins providing a high density display of antigenic sequences. The capacity to serve as carriers of antigenic sequences derived from either the parental virus or foreign sources has broadened their potential as prophylactic and therapeutic vaccine candidates. They have the ability to trigger key pathways of the immune system accessing major histocompatibility class I and II specific antigen presentation in dendritic cells.

Novel strategies to manipulate or to enhance immunogenicity by utilizing biochemically modified VLPs have been developed by changing the level of disulfide bonding and by modifying the level of glycosylation. VLPs composed of biochemically modified HBsAgS subunits are utilized to illustrate a possible general principle that extends beyond hepatitis B research and encompasses strategic developments to manipulate immune responses by using non-native immunogens to promote significant benefits to human health.

To allow cell- and tissue-specific approaches, VLPs with targeting ability were designed and generated. The recombinant VLPs are decorated with functional antibody domains that specifically target immune cells providing superior immunogenicity compared to the native (wildtype) VLPs. The presence of functional antigen-binding domains arrayed on VLPs will allow the development of universal molecular devices with specificities determined by the selected antibody domains.

Biography

Hans Netter completed his doctorate at the University of Munich, and Max-Planck Institute for Biochemistry, Germany in 1990 in the field of “autoimmunity”. In the following year, he joined Prof. John Taylor’s laboratory at Fox Chase Cancer Center, Philadelphia, USA to investigate the genome stability and helper independent infections of the hepatitis delta satellite. Research positions at the Medical School, University of

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NotesHamburg, Germany and Medical Virus Research Centre, Brisbane, Australia followed. He discovered novel avian hepatitis B viruses and non-conventional human hepatitis B viruses, which demonstrated a functional and evolutionary diversity of hepatitis B viruses. Due to these achievements, he was awarded a higher doctorate by the Medical School, University of Hamburg in 1997. In 2002, Hans Netter joined Monash University as Principal Investigator and Lecturer, with a research focus on immunogen - immune system interactions. He was awarded the Graduate Certificate in Higher Education in 2004. He has responsibilities in under- and post-graduate teaching and acts as the Deputy Assoc Dean (Research Degrees), Faculty of Medicine Nursing and Health Sciences, Monash University.

Helicobacter: then and now

BArry MArSHALL University of Western Australia, Nobel laureate

The early work on Helicobacter began with simple questions. “Were these bacteria living in the stomach?” and secondly, “How could they survive in the presence of stomach acid?” The bacteria were associated with gastritis, so tightly that the cause and effect hypothesis could not be understated. For a century, some experts had proposed that gastritis could lead to duodenal and gastric ulcer. The epidemiology also suggested that gastric cancer was also part of the picture. If gastritis could be controlled then stomach cancer could be prevented. Various paradoxes existed which were explained by the natural history of Helicobacter infection, which was unravelled by a volunteer experiment published in the April 1985 edition of the Medical Journal of Australia.

If the bacteria colonised most of the human race, how long had they been there? Were they recent, or had they been in humans for millennia? If they had been infecting almost all humans since the stone-age, did they actually have a useful purpose?

New data have revealed that Helicobacter can have a benefit in several ways, perhaps assisting the immune system to achieve the correct balance. Some of the most common human diseases affecting modern humans are due to a hyper-reactive immune system causing troublesome diseases such as asthma and allergic eczema. In some studies, these conditions are less common when a Helicobacter infection is present.

Out of curiosity, new genomic technologies have been applied to the Helicobacter genome. Amazingly, every human race carries a unique strain of Helicobacter and we can trace its origins back to determine our human origins.

Biography

In 2005 Barry J. Marshall and J. Robin Warren were awarded the Nobel Prize for Physiology or Medicine in recognition of their 1982 discovery that a bacterium, Helicobacter pylori, which affects 50% of the global population, causes one of the most common diseases of mankind, peptic ulcer disease.

Barry Marshall met Robin Warren, a pathologist interested in gastritis, during internal medicine fellowship training at Royal Perth Hospital in 1981. The pair studied the presence of spiral bacteria in association with gastritis. The following year (1982), Helicobacter pylori was cultured for the first time and they developed their hypothesis related to the bacterial cause of peptic ulcer and gastric cancer.

In 1984, at Fremantle Hospital, Marshall proved that the germ was harmful in a well-publicised self-administered experiment, in which he drank a culture of H. pylori. Persevering despite widespread skepticism, Marshall also came up with combinations of drugs that killed the bacteria and eliminated ulcers permanently.

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Notes In 1984 the World Health Organisation recognized H. pylori as the main cause of stomach cancer. Marshall and Warren’s work is acknowledged as the most significant discovery in the history of gastroenterology and is compared to the development of the polio vaccine and the eradication of smallpox.

In 1998 Marshall was made a Fellow of the Royal Society. In 2008 he was elected as a Foreign Member of the prestigious US National Academy of Science, an institution that was established in 1863 by President Abraham Lincoln.

Dr Marshall founded Ondek Pty Ltd in 2005, to develop and commercialise a novel drug and vaccine delivery platform, based on the use of genetically modified Helicobacter pylori. Dr Marshall and his research team have developed a set of proprietary technologies, referred to as the Helicobacter pylori Platform Technology (HPPT). The HPPT has potential application for oral delivery of immunotherapies, biopharmaceuticals and other drugs, and vaccines. The first product being developed at Ondek is an immunotherapy for the treatment of allergic asthma and food allergies in children.

Barry Marshall was born in Kalgoorlie in 1951 and attended Marist Brothers College in Perth from 1960-68. He completed his undergraduate medical degree at The University of Western Australia in 1974. He is married with four children and five grandchildren and lives in Shenton Park, Western Australia.

rotavirus vaccine development

SABALE rAJEnDrA nArAyAn Serum Institute of India

Serum Institute of India Ltd. (SIIL) is a private organization of Dr. Cyrus Poonawalla group. SIIL is world’s largest producer of vaccines in terms of number of doses. It is estimated that two out of every three children from about 140 countries are vaccinated by SIIL’s vaccine.

At present, Rotarix® and RotaTeq® are the only commercially available Rotavirus vaccines. Rotavirus vaccine is found promising and cost effective solution to control the rotavirus diarrhea. The present vaccines are stored and supplied at cold conditions of 2-8°C. SIIL has developed a vaccine with objective that the vaccine should be stored and transported without cold chain.

SIIL Rotavirus vaccine is live, oral, lyophilized pentavalent vaccine, comprises of five serotypes viz. G1, G2, G3, G4 and G9 and individually grown on Vero cells. All these re-assortant strains have VP7 gene of respective serotype of human strains and other ten genes of bovine (UK) Rotavirus strain. The vaccine is supplied along with 2.5 ml of diluent (citrate bicarbonate buffer) for reconstitution of lyophilized vaccine.

The stability of vaccine was studied at 2-8°C and 25°C for 36 months, 37°C for 24 months and 40°C for 6 months. The data suggested that the vaccine was stable for 36 months at 2°C to 8°C and 25°C. At 37°C and 40°C, the SIIL Rotavirus vaccine was stable for 24 months and six months respectively. This indicates that SIIL’s rotavirus vaccine is thermo-stable preparation, which may be used at ambient conditions.

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NotesBiography

In my current role as Deputy Manager (Serum Institute of India Ltd, Pune) I am involved in the planning and execution of the development of rotavirus vaccine. The vaccine is being developed for the pediatric use to prevent severe diarrhoea in infants. Vaccine Development work involves the Mammalian cell culture work, virus characterization and formulation development. The associated activities involve clinical trials and clinical research.

My past research interests included the development of Pod boarer resistant variety of Chickpea (Cicer arietium, L), via gene transformation, at the Plant Tissue Culture Division of the National Chemical Laboratory. At Mahyco Life Sciences Research Centre, I had worked to develop a pest-resistant cotton variety.

I completed both my undergraduate B.Sc. degree in the 1995 and my M.Sc. in 1997 at the University of Pune, India.

Systems for large scale manufacture of vaccines and biopharmaceuticals

JoHn PoWEr Zoetis

This presentation will provide an industrial perspective on the manufacturing platforms for commercial production of vaccines and biopharmaceuticals. While the majority of biopharmaceuticals approved for human use are monoclonal antibodies produced in mammalian expression systems, there is no “One-Size-Fits-All” solution for manufacture of biological medicines which then drives a degree of diversity in manufacturing platforms across the industry. This diversity is shaped by the inherent technical requirements of the producer-organism, the desire for improved product safety profiles, and the ever-increasing demand for cost-efficient production.

A survey of manufacturing platforms will be presented along with commentary on the technical issues encountered in their industrialisation.

Biography

John Power is a Director of Development in the Veterinary Medicine Research and Development division of Zoetis. In this role, John provides leadership towards the industrialisation of new veterinary vaccines in both the Asian and Global pipelines for new product flow. John has over 20 years post-doctoral experience in the scale-up of manufacturing systems for commercial production of vaccines and biopharmaceuticals. He has particular expertise in scale-up of microbial fermentations, cell culture systems, and associated processing for manufacture of biologicals. He holds a bachelor’s degree in Applied Science, a Masters in Biotechnology, and a PhD in Chemical Engineering awarded by the University of Queensland. He has also completed an MBA in Technology Management. John is a member of ISPE and AusBiotech. Before joining Zoetis, John held senior research positions in CSL Ltd, Bioproperties (Australia) P/L, Burns Philp, and Fort Dodge.

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Notes Strategies for expressing recombinant proteins in bacteria and their purification

BiLL MckinStry

CSIRO Manufacturing Flagship

So you want to produce a recombinant protein in bacteria? Where to start - what type of protein is it and is the biological function of your protein known? What do you require the recombinant protein for? Literature and bioinformatics reviews and website tools. Designing your protein expression construct, including the use of tags/fusion proteins to facilitate protein folding and purification. Selecting a suitable host cell to express your recombinant protein, and what the different protein expression strains do. Using a medium throughput approach we can rapidly screen protein expression, protein extraction and protein purification strategies for increasing the production of soluble recombinant proteins. By exploring different bacterial strains, types of culture media and induction conditions we are able to increase the likelihood of producing soluble proteins. What to do if your protein is insoluble. Scaling-up protein expression, and tips for optimising protein purification and down-stream processing with the view to delivering quality recombinant proteins.

Biography

Bill McKinstry is a protein biochemist with over 25 years technical expertise in the expression, purification and analytical characterisation of recombinant proteins for structural and functional studies. He has experience in academia and both the biotechnology and pharmaceutical industries. His research interests include; cytokines and their receptors; membrane associating proteins, host-pathogen interactions, viral structural proteins – self assembly and macromolecular complex formation, structural genomics and high-throughput technologies. He is the author of 38 peer-reviewed publications (including publications in Cell and Nature Structural Biology), has >1700 citations with an H-factor of 20, the inventor of 3 patents of which two have been licensed to pharmaceutical companies, and the development of downstream processing protocol for the production of a biotherapeutic recombinant protein about to commence human clinical trials. He holds Honorary Research Fellow appointments with both The University of Melbourne and Deakin University.

Improving secretion and purification of glycoproteins in the baculovirus-insect cell expression system

CinDy CHAnG The University of Queensland, Protein Expression Facility

Secretory glycoproteins are required for diverse research applications including therapeutic and vaccine development. The baculovirus-insect cell expression system is emerging as a favourable system for the production of recombinant glycoproteins due to its eukaryotic protein processing capabilities. Low secretion efficiency and downstream recovery are often the bottlenecks for this system. To improve protein secretion, varying expression conditions can be tested in a high-throughput expression platform. These parameters include different baculovirus systems (Bac-to-Bac® and flashBACGOLD™) with modified virus genome for enhanced secretion, signal peptides such as commonly used honeybee melittin signal sequence and glycoprotein gp67 signal sequence, insect cell lines (High Five™ and Sf9) and culture temperatures. Optimisation of the

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Notesupstream parameters is imperative for maximising protein yield for downstream processing. To aid in recovery, recombinant glycoproteins are often expressed with a widely used poly-histidine tag, which interacts with metal ions immobilised on a chromatography matrix. However, purifying histidine-tagged glycoproteins from spent culture supernatant presents a number of challenges. The presence of interfering components in the cell culture media hinders direct loading of spent culture supernatant onto chromatography column. Immobilised metal ions are ‘stripped’ from the purification media during sample loading, thus preventing the capture of histidine-tagged proteins. Large volumes of spent culture supernatant and endogenous host proteins can add further complexity to the recovery of secretory glycoproteins. Here we present practical solutions for overcoming challenges commonly encountered in expressing and purifying recombinant secretory glycoproteins from the baculovirus-insect cell expression system.

Biography

Ms Cindy Chang is the Operations Manager of the Protein Expression Facility at the University of Queensland. She has over 9 years of experience in recombinant protein expression, ranging from project design, molecular cloning to microbial and animal cell culture. Her diverse skill set in recombinant protein technology directs her in developing well-thought-out strategies for projects, ensuring efficiency and cost-effectiveness. With her in-depth understanding on the baculovirus-insect cell system, she has implemented the high-throughput multi-hosts expression platform within the facility. This streamlines the expression screen for multiple constructs with the aim to determine optimal parameters for large-scale protein production.

Yeast expression systems

BrEnDon MonAHAn CSIRO Manufacturing Flagship

Yeast have long served dual roles as research model systems and industrial production platforms for a wide-range of biological activities and products. Here I will present examples of utilising yeast as protein expression systems, in both academic and industrial contexts. The presentation will focus on a number of yeast species including the budding yeast Saccharomyces cerevisiae, and the methylotrophic yeast Pichia pastoris (Komagataella phaffii). For each I will describe the different strains and expression vector options available, as well as the respective advantages and disadvantages. Finally, recent trends in using yeast as a tool for the characterisation of small-molecule inhibitors of your target protein will also be discussed.

Biography

Brendon Monahan is a research scientist in the Biosciences Program at CMSE, project leader for the Cancer Therapeutics CRC, and a Lecturer in the Department of Genetics, The University of Melbourne. Brendon is a yeast and fungal molecular geneticist with a research background in epigenetics and gene regulation in various fungal species. Over the last few years at CSIRO, Brendon has been involved in the molecular genetic manipulation of yeast and fungal systems for the generation of bio-based products, along with leading the protein production team for the Cancer Therapeutics CRC.

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mammalian toolbox: Platform technology for developing human therapeutics

JEFF HoU Australian Institute for Bioengineering and Nanotechnology

Biologics are the most rapidly growing class of human therapeutics, with the most prominent class of biologics being monoclonal antibodies. Recombinant proteins are manufactured through various host cell systems ranging from bacteria to yeast to mammalian cells. The workhorse of the industry remains to be the Chinese Hamster Ovary (CHO) cell; in fact over 70% of all approved human therapeutics worldwide is manufactured in CHO cells.

Transient expression technology has gained significant interest in the field due to its speed and flexibility in providing recombinant proteins for initial proof of concept studies. This has significantly reduced timelines as well as gained further traction with the emergence of follow-on biologics. Stable expression technology continues to push the boundaries of mammalian cell culture with advances in host cell engineering and bioprocess development pushing productivities to well above a couple of grams per litre.

At the National Biologics Facility (NBF), we use a high throughput platform process to generate production cell lines for the manufacturing of recombinant proteins with a focus on monoclonal antibodies. Novel molecules identified through discovery platforms such as mAbLAB are reformatted and assessed using mammalian transient expression technology such as EpiCHO followed by design and creation of a stably producing mammalian cell line via either a ClonePix FL or flow cytometry facilitated cloning method.

Apart from cell line engineering and platform design, NBF have large scale manufacturing capabilities which includes a suite of equipment targeted at mammalian cultivation, harvesting, purification and analytics to convert basic raw materials into high-valued therapeutics. NBF’s current plant also uses a number of single-use bioreactors and continuous culture as a mode of cultivation as a way to increase productivity and reduce the environmental footprint.

Biography

Working with Prof. Peter Gray, A/Prof Stephen Mahler and Dr. Martina Jones, his part of the key leadership group at the QLD Node to support advanced biologics manufacturing in Australia. Dr. Hou’s background is in the area of mammalian host cell engineering and adapting high throughput processing to the area of biologics manufacturing. He has been involved in many industry projects taking lead candidates through CMC development and developing a path into the clinic. His current role is the Operations Manager at the National Biologics Facility (QLD Node) where his focused on developing tools for manufacturing of biologics as well as delivering a manufacturing service to help translate basic research.

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Notesdiscovery and development of novel human therapeutics using recombinant protein expression in mammalian cells

CAtHErinE oWCzArEk CSL Limited

Recombinant protein biologics such as monoclonal antibodies and coagulation factors are becoming increasingly important in today’s pharmaceutical industry. As they are secreted proteins with complex folding and post-translational modifications they need to be generated in mammalian cells. Production of these proteins for pre-clinical studies using the technique of transient gene expression is an efficient, time and cost-effective alternative to developing stable cell lines. Our current transient gene expression methods enable us to produce mg-gram quantities of proteins within 1-2 weeks from the time of generation of the appropriate protein expression construct. We have evaluated the recently developed Expi293™ expression system and compared it to our previously established platform that utilises the Freestyle 293™ system.

A combination of both transient expression technologies has allowed us to rapidly screen, identify and characterise multiple novel protein-based human therapeutic drug candidates.

Biography

Dr Catherine Owczarek leads the Recombinant Protein Expression Group located at the Bio21 Institute. After gaining a PhD at the John Curtin School of Medical Research, Canberra, Catherine completed her post-doctoral studies at the Sir William Dunn School of Pathology in Oxford, the Walter and Eliza Hall Institute and then was a Senior Research Fellow at the Monash Institute of Medical Research where she was appointed a Senior Scientist of the Institute. Since joining CSL in 2004 Catherine has directed the Research Group’s efforts in the successful development of a program to produce mammalian-derived recombinant proteins in scaleable suspension culture systems using disposable cell culture technology. She is involved in a range of early phase drug discovery campaigns where there is a high demand for recombinant proteins.

careerlounge: connecting students, employers and educators

DoMiniqUE FiSHEr CareerLounge

CareerLounge Pty Ltd develops digital solutions for students, educators and employers. CareerLounge was established in 2008 when co-founder, John Edward Collins, discovered that there was a critical need for better communication between students, educators and employers. CareerLounge offers a simple to use suite of products designed to improve and enhance decision making, communications and transactions between each of these related stakeholders.

www.comet.is and www.quillo.in have evolved from the original web application, currently known as www.careerlounge.com.au, as two separate but highly complementary products. Commercially launched in October 2012, www.careerlounge.com.au is to be re-launched as Comet (www.comet.is) and Quillo (www.quillo.in) in July 2014.

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Notes Comet is a global digital community made up of registered students, employers, service providers and educators who create and use Comet Profiles (individuals) and Comet Pages (organisations) to come together by industry sectors known as Comet Villages. Each of the [43] Villages highlight opportunities available in that sector by way of linked editorial and user generated content; and, relevant education and employment opportunities in each Village.

Quillo, a complementary product, is the career pathway wiki organized by Villages that provide users with formal industry definitions of job positions, career paths, preferred education and training options and associated services.

Biography

Dominique is the Managing Director and Executive Chairman of CareerLounge Pty Ltd (as well as one of its founders). For more than 30 years Dominique has worked in communications telecommunications and information technology; specifically in the application of technology in business and electronic commerce and the commercialisation of new technologies. Dominique is a highly experienced executive, and public and private company director and has held directorships with NRMA, Insurance Australia Group (IAG), Pacific Brands, Sydney Opera House, The Malthouse Theatre, Australia Council of the Arts, Dance Board, Prostate Cancer Foundation, Royal Hospital for Women Advisory Board Chairman, AIDS Fund Raising Trust, Communications and Media Law Assoc, was on the ICT Advisory Board to the Minister for Communications, the Hon Helen Coonan, Chairman of Sky Technologies Pty Ltd, and is Chairman of ASX listed Circadian Technologies Ltd.

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Notes

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Notes control of glycosylation profiles in cell bioprocesses

MiCHAEL BUtLEr University of Manitoba

The glycoform profile of a monoclonal antibody (Mab) determines many functional properties that affect therapeutic efficacy. Common variations of the conserved Fc glycan include galactosylation, fucosylation and sialylation. The observed glycan profile of the final product can depend upon the producer cell line, the growth media, the culture conditions as well as the Mab protein structure. Post-translational modification of proteins, particularly glycosylation is affected by culture conditions including the availability of nutrients. During fed-batch and other cell culture strategies nutrients are often maintained at low concentrations to ensure an efficiency of energy metabolism. However, there is a possibility that these strategies may cause variability in the macro- or micro-heterogeneity of glycosylation of synthesized glycoproteins. We have shown that low nutrients levels present in fed-batch cultures may alter the glycan profile as well as glycan occupancy by reducing the availability of the essential precursors during glycosylation in the Golgi.

We have been able to produce Mabs with a wide range of glycan microheterogeneity in culture by controlling the parameters of culture and by the use of several glycoprotein processing inhibitors. The modified glycosylation profiles of the Mabs were determined with HILIC (hydrophilic interaction liquid chromatography) analysis.

The presentation will review bioprocess parameters that can be controlled in order to minimize batch to batch variation of Mab glycosylation. Strategies will also be discussed to produce Mabs with pre-defined glycan structures.

Biography

Michael Butler is a Distinguished Professor at the University of Manitoba, Canada. Previous appointments include Associate Dean of Research at the University of Manitoba and Principal Lecturer in Biotechnology at Manchester Metropolitan University. He has also been a Visiting Scientist at the Massachusetts Institute of Technology (USA), Animal Virus Institue (Pirbright, UK) and the Universities of Oxford and Rio de Janeiro. He holds degrees in Chemistry and Biochemistry from the Universities of Birmingham, London and Waterloo (Canada).

His research focuses on the development of bioprocesses using mammalian cells in culture for the production of recombinant proteins, monoclonal antibodies and viral vaccines. He is particularly interested in the bioprocess conditions that can be used to control the biochemical structure of glycoproteins. He has collaborated closely with industry and in 2004 was the recipient of the Canadian national Synergy Award for University-Industry innovation. He is presently scientific director of the Canadian Network for Monoclonal antibody production (MabNet), which involves collaboration between several universities and companies in Canada. He has published 7 books on animal cell technology and >100 peer-reviewed scientific papers.

He is the founder and president of Biogro Technologies Inc., a spin-off company dedicated to serum-free media development for mammalian cell bioprocesses. He is a member of European Society Animal Cell Technology, Biochemical Society, Canadian Society of Microbiology, Canadian Society Chemical Engineering and President of the International Society for Protein Expression (PEACe). He is currently an editor of Biotechnology Advances and on the editorial board of Biotechnology and Bioengineering.

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NotesPurification and characterisation of mammalian expressed proteins: Key assessment considerations for candidate/target selection in a research based commercial environment

PiErrE SCotnEy AnD PEtEr SCHMiDt CSL Limited

The discovery and development of drug candidates in the biopharmaceutical industry involves the design, expression and purification of a large number of proteins such as therapeutic antibodies. With custom gene synthesis and high-throughput sequencing readily available to researchers, the bottleneck to the generation of larger numbers of proteins has shifted to the purification of proteins in an efficient and economical way. Therefore, despite the existence of well-established methods to purify antibodies in the range of 1 – 100 mg (e.g. AKTA Xpress platform) there is a continuous need to further miniaturise antibody purification in order to increase the throughput while reducing the associated time and cost. In order to achieve these goals we have developed a high-throughput antibody purification platform suitable to purify up to 96 antibodies a day in a time and cost-effective manner. The obtained yields are sufficient for biochemical screening technologies. When the number of candidate molecules has been narrowed down the production of drug candidates can be scaled up to obtain sufficient material for in vitro and in vivo functional assays.

When screening libraries for drug candidates, such as therapeutic antibodies, the leads are initially ranked by binding affinity to their target or potency in biochemical and cell based assays. Biopharmaceuticals have characteristics associated with the molecule being protein based which can affect the potential for the candidate to be developed into a drug. We have implemented a screening process early in the drug discovery phase that investigates the developability of lead candidate biopharmaceuticals by characterising the protein’s biochemical and biophysical properties, such as post-translational modification homogeneity, inherent and thermal stability, amenability to high concentration formulation and particle analysis. The information from developability profiling of candidate molecules early in drug discovery facilities in the selection of which lead biopharmaceuticals will progress in the development process.

Biography: pierre scotney

Dr Pierre Scotney is a Senior Scientist in Research at CSL Limited, as well as an Honorary Member of the University Melbourne at the Bio21 Institute. He has extensive experience in the pharmaceutical and biotechnology sector having worked at Roche Pharmaceuticals, Zenyth Therapeutics and CSL Limited. He completed his PhD in biochemistry at the University of Bristol (UK) in 1999 and came to Australia to undertake postdoctoral research at St. Vincent’s Institute of Medical Research (Melbourne). In 2001 he began investigating the biology of Vascular Endothelial Growth Factors (VEGFs) with an industry-partnered post-doctoral fellowship at the University of Melbourne and biotechnology company Zenyth Therapeutics (then Amrad Corporation). In 2004 he joined Zenyth to continue the research and develop novel therapeutics that targeted VEGFs. In November 2006 Zenyth Therapeutics was acquired by CSL Limited and Dr Scotney was appointed as a Senior Scientist, Research, located at the Bio21 Institute. He currently works with a large multidisciplinary team focused on the discovery and development of new recombinant antibody and protein-based medicines to treat serious human disease. Dr Scotney has published in peer-reviewed publications and has inventorship on patent filings.

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Notes Biography: peter schmidt

Peter started his scientific career at the Carl-von-Ossietzky University in Oldenburg, Germany, in the department of Neuro-Biochemistry. In the following four years he worked as PhD student at the Cardiovascular Research Centre of Bayer HealthCare in Wuppertal, Germany, in the field of NO-mediated blood pressure regulation. Based on this work, Peter was awarded an Alexander-von-Humboldt Fellowship which allowed him to move to Melbourne and to continue his research for two more years at the department of pharmacology of Monash University. After this short visit in academia he started to work for four years at CSIRO in Parkville on the expression of human influenza Neuraminidase. Since 3 years Peter is working as senior scientist in the Protein Technologies group of CSL R&D in Parkville.

Structural biology at the australian synchrotron

JoSE VArGHESE CSIRO Manufacturing Flagship

Since first light in April 2007 the Australian Synchrotron at Clayton in Victoria has ushered in a new era in biological research in Australia. Australian scientists have no longer been limited by the availability of beamtime on a few overseas synchrotrons. Over the last 7 years access to several different experimental platforms for research into the structure of biological macromolecules have been providing insights into structural biology. Two protein crystallography beamlines have been fully operational, the first from a bending magnet and is designed to be a high throughput MAD (Multiple Anomalous Diffraction) designed to exploit the use of seleno- methionine derivatives to phase X-ray data from protein crystals. The other much more intense beamline from an insertion device is designed to be able to collect diffraction data from crystals as small as 10 microns in size. Other beamlines such as SAXS (Small Angle X-ray Scattering) for determining the shapes of protein in solution, XAFS (X-ray Absorption Spectroscopy) for studying metal protein interactions are currently available for users, and a CD (Circular Dichroism) for studying protein secondary structure has been proposed.

Biography

Varghese was trained in Australia as a theoretical physicist with a background in mathematics and natural philosophy. He did his post graduate training in experimental physics and later in neutron physics. On returning to Australia he used his physical science training to study biological systems, resulting in his multi-disciplinary approach to solving problems in biology and acquiring skills in molecular and cellular biology. This has enabled him to form and lead multidisciplinary teams of scientists that are making inroads into complex biological problems. The impact of his work in influenza is now being realized globally with emerging pandemics. Recently he was leading the development of drugs and diagnostics for Alzheimer’s disease and mental disorders in the P-Health Flagship. He has also acted in advisory roles for State and Federal Governments in areas of scientific infrastructure in particular the Australian Synchrotron where he led the construction of the two protein crystallography beamlines. He is currently a CSIRO Retired Fellow.

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NotesSimple and low cost quality control of expressed proteins by mass spectrometry

niCHoLAS A. WiLLiAMSon The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne

Protein expression has become a key technology for biomedical research. The integrity and quality of the final expressed product is often critical to the sucess of the downstream experiments for which proteins were produced. Many people simply use SDS PAGE analysis to check their expression products. This talk, however, will discuss the inexpensive and straightforward use of mass spectrometry to explicitly determine the primary structure of the expressed products. Examples will be shown that highlight the ability of mass spectrometry to identify point mutations, truncations and modifications in recombinant products that would not be detected by SDS PAGE. Examples of protein complex formation and monitoring refolding experiments will also be shown. The key point of this presentation is to show that mass analysis of expressed proteins is fast, simple, cheap and can easily be made a routine quality control step for expressed protein work.

Biography

Dr Nicholas Williamson is the manager of the Proteomics and Mass Spectrometry Platform (MSPF) at the Bio21 Institute at the University of Melbourne. He has more than 20 years experience in proteomic mass spectrometry and is an expert in the primary and secondary structure analysis of proteins and peptides. He now runs the Proteomics Core facility which provides a unique, open-access mass spectrometry resource to research groups located in Melbourne. The facility offers users highly subsidised direct access to instrumentation and the expert training required to perform their own proteomic research.

Physical techniques in a protein workflow

SHAnE SEABrook CSIRO Manufacturing Flagship

There is no single method that allows complete characterisation of a bio-macromolecule and its various interactions. The best insight is gained when you can access as many techniques as possible, making sure to develop a robust understanding of your protein and how it responds to its immediate environment (its purification or storage formulation) and with ligands of interest (co-factors, drug candidates).

At CSIRO we routinely use thermal stability and spectroscopic techniques to help us figure out how a protein behaves – by building multidimensional insight we can better understand the impact the chemical and physical environment will have on our target of interest. Most importantly, we clearly outline our expectations from an assay; are we trying to develop a comprehensive understanding of the protein

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Notes behaviour, are we trying to make a protein more suitable for downstream analysis, or are we screening for “hit” conditions, such as ligand binding assays and formulation screening?

This seminar will summarize some of the techniques available and where they might fit in to your experiment workflow, how you can make decisions based on the data you’ve collected, and the alternative strategies that may be available.

Reference: Seabrook, S. A.; Newman, J., High-Throughput Thermal Scanning for Protein Stability: Making a Good Technique More Robust. ACS Combinatorial Science 2013.

Biography

Shane completed his PhD (physical chemistry) at the University of Sydney, followed by post-doctoral projects in France and the Netherlands on macromolecular and biopolymer characterisation. Shane joined the biophysics in the CSIRO Materials Science and Engineering division in late 2009, and has been working within the Collaborative Crystallisation Centre (www.csiro.au/c3) developing methods that aid structural biology. Shane is currently completing an MBA at the Melbourne Business School.

australian therapeutics pipeline

StEWArt HAy Therapeutic Innovation Australia

Therapeutic Innovation Australia is a not for profit organisation tasked with implementing an Australian Government research infrastructure project. A key objective for TIA with this project is to improve the efficiency and effectiveness of translational health research in Australia. This presentation will briefly outline TIAs research data gathered over the last twelve months and it will describe the implementation of TIAs strategy to enable better translation of health discoveries into clinical application.

TIA is, amongst its 18 investments, supporting protein production capabilities through development of the National Biopharmaceutical Facility, a collaborative between the University of Queensland and CSIRO.

Biography

Stewart Hay has a diverse background in the science industry having worked in medical research, and the pharmaceutical, nutraceutical, biofuel and medical device industries. He holds a PhD in the field of cancer cell biology and has conducted research into diabetes, Langerhans Cell Histiocytosis, the characterisation of a novel viral particle, antibody production and mechanisms of apoptosis. Over the last decade Stewart has held various management roles leading to his appointment as Chief Executive Officer at Therapeutic Innovation Australia. In this capacity Stewart manages the provision of funding for development of translational research facilities and several national advisory committees.

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Notesthe National Biologics Facility (NBF) – a joint aIBN/cSIro facility

PEtEr GrAy Institute for Bioengineering and Nanotechnology (AIBN), The University of Queensland

Biologics are a class of human therapeutics comprised of proteins such as monoclonal antibodies produced by recombinant DNA technology. The specificity and other advantages of biologics has meant that they have rapidly gained acceptance, eg: seven out of the world’s top ten selling drugs are now biologics; biologics now account for 17% of the total sales of pharmaceuticals; and annual sales of biologics are growing at 20% per annum, compared to 7% for traditional small molecule pharmaceuticals.

For the above reasons, the NCRIS (National Collaborative Research Infrastructure Scheme) Roadmap of February 2006 provided funding to establish national research infrastructure in the area of Biologics. Following a review in 2010, this national research infrastructure was consolidated into the ‘National Biologics Facility’ (NBF) consisting of two nodes, one located at the AIBN in Brisbane, and the other located at the CSIRO in Melbourne. Since 2006, the two nodes of the NBF have attracted total funding of over $28 million from users, NCRIS (2006), EIF/SSI, NCRIS (2013) TIA, Queensland and Victorian State Governments, and CSIRO and AIBN/UQ sources.

Currently NBF has 22 staff working in the two nodes. The NBF has gained an excellent reputation with the Australian research community for the high quality of its work and services. Over the last five years the NBF has completed over 500 projects for a number of SME’s and public sector research organisations, and has produced and purified over 10,000 litres of mammalian cell cultures, and over a kilogram of purified protein. The projects carried out include mammalian cell-line development, bioprocess development, monoclonal antibody and vaccine isolation and production, and the production of pre-clinical amounts of biologics for animal and other testing.

Biography

Professor Peter Gray was appointed the inaugural director of the Australian Institute of Bioengineering and Nanotechnology (AIBN) at the University of Queensland in 2003.

Previously he was Professor and Head of Biotechnology at UNSW and Director of the Bioengineering Centre, and a Senior Principal Research Fellow at the Garvan Institute of Medical Research in Sydney.

Professor Gray has had commercial experience in the USA working for Eli Lilly and Co and for the Cetus Corporation as well as previously holding academic positions at University College London, and at the University of California, Berkeley.

His research interests are focussed on engineering mammalian cells to produce the complex proteins called biologics which are gaining rapid acceptance as human therapeutics, and on developing human stem cells bioprocesses suitable for clinical application.

Professor Gray was one of the founders and is a past President of the Australian Biotechnology Association, AusBiotech. He is a Fellow of the Australian Academy of Technological Sciences and Engineering (ATSE) and of the Australian Institute of Company Directors, and has been named as one of Australia’s 100 Most Influential Engineers. Awarded the Centenary Medal by the Australian Government in 2001.

He is a Vice-President of ATSE, and serves on the Boards of Biopharmaceuticals Australia Pty Ltd, ACYTE Biotechnology Pty Ltd, Stem Cells Ltd, ECI Inc, New York, and a number of State and Federal Government Councils and Committees.

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Notes BPa and the biologics facility of the future

BrEtt WHitECroSS BioPharmaceuticals Australia

BioPharmaceuticals Australia (BPA) was established to support the country¹s biopharmaceutical development industry and fill a critical national capability gap identified by both the Commonwealth and QLD State Governments GMP mammalian cell product contract manufacturing. In October 2013, as a result of collaboration between Government, industry, philanthropy, and the research sector, a $65M biologics facility was opened in Brisbane. A partnership between BPA and Patheon Biologics (formerly DSM Biologics) has seen the multinational CMO establish a subsidiary in Australia and operate the world-class ³biologics-facility-of-the-future².

BPA continues to support this collaboration and strengthen the local industry through the Biopharmaceutical Development Fund (BDF) designed to assist not-for-profit organisations and fledgling biotech companies access this new national capability, while attracting new developers to Australia.

Biography

Brett Whitecross has 8 years¹ experience in the biotechnology sector, working at the interface between the public and private sectors and in a variety of capacities. As Business Development Manager for BioPharmaceuticals Australia (BPA) Brett has been involved in the establishment of a world-class biologics manufacturing facility for mammalian cell products, the attraction of a multinational Contract Manufacturing Organisation to Australia as BPA¹s operating partner, and biopharma industry development. Brett is continuing with BPA¹s strategic initiatives, working with a global focus to attract clients to the new facility.

Previously, Brett has held business development positions with PharmaSynth and AusBiotech, has worked in policy development for the Government, and currently serves as a committee member of AusBiotech¹s Queensland Branch.

Brett has received a Bachelor of Biotechnology Innovation with honours from the Queensland University of Technology (QUT) and an MBA from the University of Queensland (UQ).

the Victorian antibody Initiative (VaI)

CAroLinE LAVErty Monash Antibody Technologies Facility

A joint initiative between the Walter and Eliza Hall Institute (WEHI) and Monash Antibody Technologies Facility (MATF) now provides the biomedical research community with an antibody partner fully attuned to the demands of world-class science.

The Victorian Antibody Initiative (VAI) brings together the state-of-the-art automated production facilities at MATF with the premier customised development services at WEHI for the very best in monoclonal and polyclonal antibody production.

For more than two decades, the Walter and Eliza Hall Institute’s Antibody Facility has been integral to the institute’s leading position in biomedical research and developing

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Notespre-clinical drug candidates and has earned a reputation for providing world-class antibody development services. The personalised approach to customer service guarantees that academic and industry users return to this facility time and again for their antibody requirements.

In 2008, investment by Monash University, the Victorian State Government and the Australian Federal Government (Bioplatforms Australia) created scale through the Monash Antibody Technologies Facility, one of the most sophisticated, high-throughput monoclonal antibody facilities in the world.

In partnership, two of Australia’s leading and complementing antibody facilities can provide you with a “one stop” point of contact to access the best technology and customer service for production of your immunological tools and reagents.

The Victorian Antibody Initiative is lead by a team having exceptional experience in antibody design and production, including Kaye Wycherley and Paul Masendycz from The Walter and Eliza Hall Institute Mark Sleeman and Caroline Laverty from Monash University.

Biography

I studied at University College London (UCL) to obtain my PhD in physical-organic chemistry under Prof. Mike Abraham. After which I moved to Pfizer as a Team Leader in the Drug Metabolism Automation Team where we were responsible for the development of a fully automated high throughput screening system for thousands of novel drug compounds. As part of an exciting life change, I moved to Australia and joined Monash University. Firstly at the Center for Drug Candidate Optimisation for 2 years and then the challenge of getting a fully automated facility for the production of monoclonal antibodies up and running brought me to MATF in 2009.

GmP considerations for recombinant protein manufacture and working with a cmo

BrEtt BUtCHEr PharmaSynth

TRecombinant proteins can be produced in a diverse array of microbial and mammalian cell lines. There are many considerations and decisions to be made as you take your recombinant protein from the laboratory benchtop into the clinic including developing manufacturing processes that are amenable to cGMP, include an element of future proofing and produce the optimum yield to ensure cost effective manufacturing. Once you have made the decision to outsource your manufacturing, the importance of selecting the right CMO cannot be understated as:

• Biologics manufacturing is a technologically complex, highly regulated process.

• Large complex protein structures are unstable and have a low tolerance for error.

• Even the best product can fail if it experiences substantial delays in the development process.

• There is a time commitment involved in transferring the technology into the CMO required to make your recombinant protein.

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Notes Biography

Brett Butcher has worked for Progen Pharmaceuticals since 2002, performing the role of Senior Scientist, specialising in the manufacturing and downstream processing of pharmaceuticals. In 2009 he became the Production Manager of PharmaSynth and coordinated Progen’s anti cancer drug PI-88s progression from preclinical to phase 3 manufacture. He has also been responsible for the tech transfer and manufacture of many client products for both clinical applications and marketed products. Brett holds a PhD in yeast biochemistry which investigated real time membrane fluidity modulation as part of the adaptative response of yeast.

Insulin receptor: Production of midi-, mini- and micro-constructs: targeted design of insulin receptor for crystallography of Site 1

JoHn MEntinG Walter and Eliza Hall Institute of Medical Research

Investigating the structural biology of the human insulin receptor brings challenges both in terms of protein production and in terms of crystallography. We have utilized two constructs of the human insulin receptor using stably-transfected CHO Lec8 cells. The first produces a thrombin-cleavable form of the three-domain L1-CR-L2 described as a “minireceptor”. Thrombin cleavage yields the two domain L1-CR “micro-receptor” that is capable of binding insulin in the presence of the receptor’s so-called alphaCT peptide. A further construct, consisting of a homodimer of a L1-CR-L2-(FnIII-1)-alphaCT receptor polypeptide (a “midi-receptor”) was produced and purified by insulin-affinity and size-exclusion chromatography. The micro- and midi-receptor constructs yield crystals in CSIRO C3 screens and their structures have been solved by molecular replacement. Despite crystallographic limitations, a consequence of protein heterogeneity, these structures are the first to reveal the way in which insulin binds to its primary receptor binding site and the manner in which the insulin B-chain C-terminal segment is displaced from the hormone core upon receptor engagement.

Biography

John Menting completed a B.Sc.(Hons) at the University of Melbourne in 1986 with Professor Gerhard Schreiber studying the transport of thyroid hormones across the blood-brain barrier. He completed a Ph.D. at La Trobe University with Professor Bob Scopes working on a plant cytochrome P450 related to the “blue rose” project of Calgene Pacific (now Florigene) in 1993 and was a member of the team that isolated and patented the “blue gene”.

In two postdoctoral fellowships, first at WEHI with Professor Alan Cowman and then at the Monash Microbiology Department with Professor Ross Coppel, he studied drug resistance in malaria and the merozoite surface protein MSP4. He then joined the Structural Biology Division at WEHI and worked with Dr. Jacqui Gulbis on translocases of the mitochondrial outer membrane until in 2008 he changed course and established the laboratory of Associate Professor Mike Lawrence shortly after Mike moved to WEHI from the CSIRO at Parkville. Since then he has continued the investigation of the insulin receptor and insulin-like growth factor receptor 1 leading to the publication in 2013 of the first structures of insulin bound to Site 1 of the insulin receptor.

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Notestelomerase: a promising target for cancer

SCott CoHEn Children’s Medical Research Institute

Telomeres, the repetitive DNA-protein complexes at the ends of linear chromosomes, shorten with each cycle of DNA replication, providing a counting mechanism to limit the number of times a cell can divide. Most cancer cells have activated the ribonucleoprotein enzyme telomerase to add telomeric DNA repeats and thereby counteract telomere shortening, allowing for unlimited proliferation; in contrast, normal cells have undetectable or low levels of telomerase. Inhibition of telomerase is therefore a promising avenue for future anticancer therapy that should be effective against a broad range of cancers while displaying few side effects.

This talk will begin with a brief overview of telomere and telomerase biology, and then cover the journey from an enzyme complex of unknown composition through its purification, identification of components, and development of an overexpression system – research that from the outset has involved collaboration with the Fermentation Lab at CSIRO Materials Science & Engineering. The presentation will conclude with progress towards a low-resolution EM structure of the human telomerase enzyme complex.

Biography

Dr. Scott Cohen received a PhD in Chemistry from the California Institute of Technology, elucidating the molecular mechanism of the “enediyne” class of naturally-occurring DNA-cleaving agents. From Caltech he moved to the laboratory of Professor Tom Cech at the University of Colorado to study the mechanism and conformational dynamics of large catalytic RNA molecules (ribozymes). In 2003 he began his research into telomerase at Children’s Medical Research Institute (Westmead NSW), and in 2007 established the composition of the human telomerase enzyme complex, reported in Science. Dr Cohen’s singular research aim is telomerase structure.

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DAy tHrEE – HAnDS onWorkshops

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Notes

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Durga Acharya CSIRO [email protected]

Campbell Aitken CSL Limited [email protected]

Layla Alhasan RMIT University [email protected]

Hameeda Al-Musawi BMS [email protected]

Jess Andrade CSIRO [email protected]

Main Ern Ang University of Adelaide [email protected]

David Antonjuk BioNovus Life Sciences [email protected]

Thilini Anupama University of Melbourne [email protected]

Tracee Archibald GE Healthcare Life Sciences

Michelle Audsley Monash University [email protected]

Samar Babkair Burnet Instiute [email protected]

Matt Banfield Sigma Aldrich [email protected]

Paul Barrett ATA Scientific [email protected]

Sue Barrett CSIRO [email protected]

Rebekah Bernard RMIT University [email protected]

Kanika Bhadoria Sartorius [email protected]

Elham Bidram University of Melbourne [email protected]

Jamie Black CSL Limited [email protected]

Melissa Blues Merck Millipore [email protected]

Violeta Bogdanoska Eppendorf [email protected]

Mark Bourke Labtek [email protected]

Kerry Breheney CSIRO [email protected]

Tracy Bryan Children’s Medical Research Institute [email protected]

Ashlee Burt Burnet Institute [email protected]

Brett Butcher PharmaSynth [email protected]

Rebecca Butcher CSL [email protected]

Michael Butler University of Manitoba [email protected]

Avinesh Byreddy Deakin University [email protected]

Steven Byrne CSL Limited [email protected]

Teresa Carvalho Monash University [email protected]

Cindy Chang The University of Queensland [email protected]

Zhian Chen Monash University [email protected]

Chris Chiu Walter and Eliza Hall Institute of Medical Research [email protected]

Jocelyn Choo Monash University [email protected]

Seong Hoong Chow Monash University [email protected]

Scott Cohen Children’s Medical Research Institute [email protected]

Peter Collins CareerLounge [email protected]

Richard Cormick 3M Purification [email protected]

David Cossens GE Healthcare Life Sciences [email protected]

Helen Dacres CSIRO [email protected]

Emma Dal Maso Monash University [email protected]

Greg D’Cruz Box Hill Institute g.d’[email protected]

Stephen de Lacey Eppendorf [email protected]

Matthew Dennis Monash University [email protected]

Pathum Dhanapala CSIRO [email protected]

PArticiPAntS liSt

32


Alison Digney Life Technologies [email protected]

Esin Donaldson CSL Limited [email protected]

Theresa Downey CSIRO [email protected]

Valentina Dubljevic Patrys Limited [email protected]

Geoff Dumsday CSIRO [email protected]

Sarah Edwards Monash University & CSIRO AAHL [email protected]

Heidi EL-Tahhan CSL [email protected]

Rochelle Enright Genesearch [email protected]

Lou Fabri CSL [email protected]

Friederike Fehr CSIRO [email protected]

Dominique Fisher CareerLounge [email protected]

Chwan-Hong Foo CSIRO [email protected]

Mike Gerometta Circadian Technologies Ltd [email protected]

Veronica Glattauer CSIRO [email protected]

Benjamin Gloria Ludwig Institute for Cancer Research [email protected]

Christopher Gonelli University of Melbourne [email protected]

Julian Grusovin CSIRO [email protected]

Jun Gu Burnet Institute [email protected]

Christoph Hagemeyer Baker IDI Heart and Diabetes Institute [email protected]

Joshua Hardy Monash University [email protected]

Natalie Hayes Eppendorf [email protected]

Thomas Hayman WEHI [email protected]

Jack Hertzog Monash [email protected]

Behnaz Heydarchi University of Melbourne [email protected]

Anita Hill CSIRO [email protected]

Jeremy Hoglin VWR International [email protected]

Jeff Hou University of Queensland, AIBN [email protected]

Linda Howell CSIRO [email protected]

Jon Ince GE Healthcare Life Sciences [email protected]

Amelia Jalanski CSL Limited [email protected]

Edwina Jap Baker IDI [email protected]

Anthony Jay PharmaSynth [email protected]

Carina Joe RMIT University [email protected]

Marietta John CSIRO [email protected]

Martina Jones The University of Queensland [email protected]

Michael Keir Lonza Australia Pty Ltd [email protected]

Sadia Nawroz Khan Monash University [email protected]

Keith Khoo CSIRO/Deakin [email protected]

Tum Khwanmuang ENFA Pty Ltd [email protected]

Natalie Kikidopoulos RMIT University [email protected]

Anastassija Konash Universal Biosensors [email protected]

Mellisa Kowalski GE Healthcare Life Sciences

Mylinh La CSIRO [email protected]

Samantha Lagaida University of Melbourne [email protected]

Caroline Laverty MATF [email protected]

Rachel Lee Monash University [email protected]

33


Jo Levett Sartorius [email protected]

Yulia Losev Baker IDI Heart and Diabetes Institute [email protected]

George Lovrecz CSIRO [email protected]

Louis Lu CSIRO [email protected]

Jasmina Luczo CSIRO [email protected]

Therese Lynch CSL Limited [email protected]

Siti Nurfatimah M.Shahpudin Monash University [email protected]

Mayra Machuca Monash University [email protected]

Luis Malaver Malaver Ortega CSIRO [email protected]

Kirstie Mangas University of Melbourne [email protected]

Ashley Marsh Murdoch Childrens Research Institute [email protected]

Barry Marshall University of Western Austrlia [email protected]

Kelly McDonagh GRACE [email protected]

Bill McKinstry CSIRO [email protected]

James McRae 3M Purification [email protected]

Bronwyn Meehan University of Melbourne [email protected]

John Menting Walter and Eliza Hall Institute for Medical Research [email protected]

Daryn Metti Bioscientific pty ltd [email protected]

Ann Mills POCD Scientific [email protected]

Oskar Mills Private [email protected]

Joyanta Kumer Modak Monash University [email protected]

Brendon Monahan CSIRO [email protected]

Naomi Morison Life Technologies [email protected]

Jacqueline Morris CSIRO Australian Animal Health Laboratory [email protected]

Bevan Morton Thermo Fisher Scientific [email protected]

Matthias Nachtschatt CSIRO [email protected]

Musammat Nahar University of Melbourne [email protected]

Nishen Naidoo Bio-rad [email protected]

Amy Nankervis Pall Australia [email protected]

Vani Narasimhulu Burnet Institute [email protected]

Hans Netter Monash University [email protected]

Aaron Ng 3M Purification [email protected]

Liza Nguyen Bristol-Myers Squibb [email protected]

Julie Nigro CSIRO [email protected]

Amir Noormohammadi University of Melbourne [email protected]

Gail O’Connell In Vitro Technologies [email protected]

Catherine Osborne Thermo Fisher Scientific [email protected]

Catherine Owczarek CSL Limited [email protected]

Lorien Parker St. Vincents Institute [email protected]

Paresh Parmar CSIRO [email protected]

Timothy Patton Burnet Institute [email protected]

Hannah Pearce Baker Heart Research Institute [email protected]

Lesley Pearce CSIRO [email protected]

34


Grant Peck CSIRO [email protected]

Yong Peng CSIRO [email protected]

Angelo Perani LICR [email protected]

Jessica Petracca CSL Limited [email protected]

Tam Pham CSIRO [email protected]

Tram Phan CSIRO [email protected]

Pat Pilling CSIRO [email protected]

Luisa Pontes-Braz CSIRO [email protected]

JOHN POWER ZOETIS [email protected]

Rasika Premaratne Monash University [email protected]

Tam Quach CSL Behring [email protected]

Adam Quek Monash University [email protected]

Rebecca Rabi Monash University [email protected]

Soizic Radulovic CSIRO [email protected]

Anna Raicevic CSIRO [email protected]

John Ramshaw CSIRO [email protected]

Ali Razaghi James Cook University, Australia [email protected]

Peter Regan Kinesis Australia [email protected]

Howard Roberts Millennium Science [email protected]

Chiara Ruggeri Monash university [email protected]

Rajendra Sabale Serum Institute of India Ltd [email protected]

Abu Iftiaf Md. Salah Ud-Din Monash University [email protected]

Bansi Sanghvi Bio-Rad Laboratories [email protected]

Paul Savage CSIRO [email protected]

Peter Schmidt CSL [email protected]

Judy Scoble CSIRO [email protected]

Pierre Scotney CSL Limited [email protected]

Shane Seabrook CSIRO [email protected]

Michiel Selen TrendBio Pty Ltd [email protected]

Kaylene Selleck CSIRO [email protected]

Madhu Sharma Sengupta Monash University [email protected]

Brian Shiell CSIRO [email protected]

sean smith Omni Executive [email protected]

Tim Smith A&D Australia C/- Labtek [email protected]

Vishal Srivastava Knoesis Biotech [email protected]

Stef Stefanakos GRACE [email protected]

Randy Suryadinata CSIRO [email protected]

Yuzhe Tang University of Adelaide [email protected]

Han Tanwar CSIRO [email protected]

Nick Tartaris VWR International [email protected]

Anais Tatry Ludwig Institute for Cancer Research [email protected]

Jan Tennent Biomedical Research Victoria [email protected]

Stephen Tibb 3M Purification [email protected]

Tak Tiong Assay Matrix Pty Ltd [email protected]

Mary Triantis bioscientific pty ltd [email protected]

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Nick Tsichlis Lonza [email protected]

Jaana Tuominen Walter and Eliza Hall Institute [email protected]

Birgit Unterweger Monash University/CSIRO [email protected]

Jose Varghese CSIRO [email protected]

Gina Vello GRACE [email protected]

Trent Warburton TrendBio Pty Ltd [email protected]

Andri Wardiana The University of Queensland [email protected]

Thomas Watts Monash University [email protected]

Christopher Weir Walter and Eliza Hall Institute [email protected]

Jerome Werkmeister CSIRO [email protected]

Brett Whitecross BioPharmaceuticals Australia (BPA) [email protected]

Eden Whitlock CSIRO [email protected]

Deborah Williams Pall [email protected]

Nicholas Williamson University of Melbourne [email protected]

Tom Wilson CSIRO [email protected]

Elton Wong Monash Institute of Pharmaceutical Sciences [email protected]

Janice Woods Millennium Science [email protected]

Amanda Woon Monash University [email protected]

Guojie Wu Monash University [email protected]

Xiaowen Xiao CSIRO [email protected]

Li Yang Monash University [email protected]

Wei Yang Monash University [email protected]

Kuok Yap ARC Centre of Excellence in Plant Cell Walls [email protected]

Mike Yarski Millennium Science Pty Ltd [email protected]

Bonnibel Yee Genesearch Pty Ltd [email protected]

Rima Youil Private [email protected]

Ling Zhu University of Melbourne [email protected]

Rick Zolzinski Merck Millipore [email protected]

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Recombinant Protein Production Facility

About the RPPFThe RPPF provides access to state-of-the-art laboratories and expertise relating to the production, processing, development and optimisation of recombinant proteins. The facility is based in Melbourne and is supported by Australia’s Super Science Initiative.

One of the challenges of bringing research and development to a commercial reality is the production of potentially valuable proteins in quantities large enough to support pre-commercial investigations and trials.

CSIRO is Australia’s national science agency. We employ 6500 people and are ranked in the top 1% of research organisations in the world. Our inventions include the Relenza flu treatment and fast WLAN. Our Recombinant Protein Production Facility (RPPF) is an important part of our research capability – helping to facilitate life-changing research across the globe.

Dr George Lovrecz at CSIRO’s Recombinant Protein Production Facility.

The RPPF is supported by the expert capabilities of various molecular and cell biology groups. We can facilitate the optimisation, scaling-up, production and purification of recombinant proteins in large quantities: from hundreds of milligrams to kilograms.

Our expertiseOur capabilities include a proven track record in fermentation and downstream processing of recombinant proteins, expressed in eukaryotic and prokaryotic hosts, such as mammalian, insect, yeast and bacterial cells.

Recent projects included the:

◆ production of monoclonal antibodies

◆ receptor, signalling and cytokine proteins

◆ bacterial and insect cell proteins

◆ various enzymes

◆ food additivies.

The RPPF provides national access to process development, optimisation and protein production in line with best-practice standards including ISO9011 certification for tissue culture work.

MANUFACTURING FLAGSHIPwww.csiro.au

Case Study: Antibodies for developing countries

In 2012 CSIRO was contracted by Program for Appropriate Technology in Health (PATH), an international not-for-profit organisation, to produce antibodies on a large scale to aid the development of new, safe, affordable and effective vaccines against rotavirus.

Rotavirus kills nearly half a million children every year, 85 per cent of whom live in developing countries. PATH is working to increase access to and effectiveness of existing commercial rotavirus vaccines worldwide.

CSIRO used their recombinant protein production facility for the large-scale ISO9001 production of six antibodies that will be used to test the quality of the rotavirus vaccines being developed.

“This unique, state-of-the-art facility allowed us to produce enough antibodies to supply PATH’s partners for the next five years,” said Prof Lovrecz.

The antibodies, developed by international researchers including the Murdoch Children’s Research Institute, will be used by PATH’s manufacturing partners in China, Brazil, and India and by the United States’ National Institutes of Health.

Researchers controlling CSIRO’s Brunswick reactor.

CONTACT USt 1300 363 400 +61 3 9545 2176 e [email protected] w www.csiro.au

FOR FURTHER INFORMATION

Manufacturing FlagshipProf George Lovrecz t +61 3 9662 7348 e [email protected] w www.csiro.au

YOUR CSIROAustralia is founding its future on science and innovation. Its national science agency, CSIRO, is a powerhouse of ideas, technologies and skills for building prosperity, growth, health and sustainability. It serves governments, industries, business and communities across the nation.

Dr Louis Lu working in CSIRO’s Recombinant Protein Production Facility.

FacilitiesThe state-of-the-art fermentation laboratories are equipped with a wide range of cell culture and fermentation equipment including:

◆ and large-scale protein production

◆ single use bioreactors up to 25L working volumes

◆ roller bottle apparatus, spinner and shaker flasks suitable for scale-up and process development work

◆ a wide variety of analytical equipment to follow cell growth and metabolism and characterise proteins

◆ downstream and purification equipment suitable to process up 500L batches of bacterial and mammalian cell culture harvests.

The RPPF is supported by the expert capabilities of various molecular and

cell biology groups to allow cell line development, protein chemistry for rapid purification and characterisation of proteins. Our direct connection to the C3 crystallisation laboratories and to the Australian Synchrotron to also allows structural studies.

Collaboration and fundingThe Australian Government, State Government of Victoria and CSIRO have invested in this facility to enhance Australia’s capacity to produce recombinant proteins in pre-commercial quantities.

The RPPF closely collaborates with The University of Queensland’s Australian Institute for Bioengineering and Nanotechnology, the Monash and WEHI Monoclonal Antibody Facilities.

ContaCt ust 1300 363 400 +61 3 9545 2176 e [email protected] w www.csiro.au

Your CsIroAustralia is founding its future on science and innovation. Its national science agency, CSIRO, is a powerhouse of ideas, technologies and skills for building prosperity, growth, health and sustainability. It serves governments, industries, business and communities across the nation.

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