New Trends in shaking: Improved culture results with new vessels Dr. Daniel Brücher, Product Manager Infors AG, Switzerland

Topics: Summary previous webinar Introduction to Thomson flasks TPP centrifuge tubes Process optimization

Nobel price: antibiotic Streptomycin (USA) 1861 1900 1949 1967 2005 Better growth using a shaker Bacterial fermentation Static Hawrylenko patent magnetic drive

Oxygen supply is a critical factor! Oxygen transfer gasphase – liquid phase Oxygen concentration in air: 21% = 300 mg/L (norm.) O2 CO2 Gas-phase  on the LIQUID SURFACE!!! Funghi Bacteria Liquid-phase Yeast Mammalian cells Max. oxygen solubility in water: Max. 7 mg/L (37°) nutrients waste Oxygen supply is a critical factor!

Function of shaking Suspend cells Mixing of the liquid Microbes and cells LOVE shaking!!! Function of shaking Suspend cells Mixing of the liquid Resolve gradients Increase surface: Enhance gas exchange! O2 CO2

x2 Influence factors oxygen transfer: Surface to volume ratio Large surface per volume: better gas transfer x2 O2 CO2 O2 CO2 Erlenmeyer flask Fernbach flask

Filling volume Very important factor with Erlenmeyer flasks: Max. 30% (250 ml total  83 ml) Optimum 25% (250 ml total  62 ml), sometimes 10% Price to pay  less capacity

Shape of the flask Very important factor with Erlenmeyer flasks: Angled sides reduce liquid movement.

„How can I get a better culture result in Erlenemeyer flasks? Use 10% - 30% max filling volume in Erlenmeyer new designs  up to 50% Increase shaking speed as much as possible Limit „cell“: shear stress Limit „bact“: flask fixing, spilling Use 50 mm shaking throw for large flasks, especially cell Consider other culture vessels!

Topics: Summary previous webinar Introduction to Thomson flasks TPP centrifuge tubes Process optimization

Bacterial applications: Ultra Yield FlasksTM and AirOTopTM Seal, Thomson Instrument Company, disposable/re-usable 2.5 L total volume 1.0 L filling volume 26.11.2018

Efficient sterile filter  Good gas exchange What´s the difference? Large-diameter neck Steep side walls  Large surface/vol ratio Baffles  Good mixing Culture protocols/application notes 26.11.2018

Efficient sterile filter/Large-diameter neck 26.11.2018

Why do baffles make a difference?  They provide good mixing  good oxygen transfer Recommended shaking speed 2.5 L: 250 rpm (25 mm, bacterial applications) 26.11.2018

7.8 L 8 x 2,5 L Ultra Yield (capacity: 12-13) Erlenmeyer: 13 x 2000 per deck 30% filling: 26 L 10% filling: 7.8 L 7 x 5 L Optimum Growth 26.11.2018

6 x 2,5 L Ultra Yield flasks Clamps: ATM 2000 26.11.2018

26.11.2018

Bacterial applications : Ultra Yield FlasksTM and AirOTopTM Seal, Thomson Instrument Company, disposable Tested by: Genentec, GSK, Pfizer 26.11.2018

Bacterial applications : Ultra Yield FlasksTM and AirOTopTM Seal, Thomson Instrument Company, disposable Tested by: Genentec, GSK, Pfizer 26.11.2018

Cell-applications: Optimum Growth FlasksTM, Thomson Instrument Company, disposable 26.11.2018

Cell-applications: Optimum Growth FlasksTM, Thomson Instrument Company, disposable 26.11.2018

Cell-applications 26.11.2018

26.11.2018

Cell-applications 26.11.2018

Field report London: New Brunswick I26. Thomson Ultra Yield. Flasks 2 Field report London: New Brunswick I26 Thomson Ultra Yield Flasks 2.5 litre 25 mm 250 rpm Bact. application „However the I26R are a complete nightmare….I am trying to return the NBS shakers, as the company cannot get the shaker to work with the Ultra -Yield flasks“ 26.11.2018

Field report London: Multitron. Ultra Yield Flasks, 2 Field report London: Multitron Ultra Yield Flasks, 2.5 litre 3deck Multitron 25 mm Number of flasks: 12 Filling volume: 1 litre Shaking speed: 250rpm (up to 300 OK as well) 26.11.2018

Recommended shaking speed: 250 rpm (25 mm) Maximum: 300 rpm (25 mm) 6 x 2,5 L Ultra Yield flasks Clamps: ATM 2000 Recommended shaking speed: 250 rpm (25 mm) Maximum: 300 rpm (25 mm) 26.11.2018

Topics: Summary previous webinar Introduction to Thomson flasks TPP centrifuge tubes Process optimization

Application: centrifuge tubes „TubeSpin® bioreactor“

Application: centrifuge tubes „TubeSpin® bioreactor“

Application: centrifuge tubes „TubeSpin® bioreactor“

Application: centrifuge tubes „TubeSpin® bioreactor“

Application: centrifuge tubes „TubeSpin® bioreactor“ Always 50 mm throw! 150 – 250 rpm (50mm)!

Shaking speed 20mL 50mm 150rpm 20mL 50mm 240rpm

Topics: Summary previous webinar Introduction to Thomson flasks TPP centrifuge tubes Process optimization

Bacterial applications : Ultra Yield FlasksTM and AirOTopTM Seal, Thomson Instrument Company, disposable Tested by: Genentec, GSK, Pfizer 26.11.2018

Increase limiting factor  better growth Optimize: always look at the WHOLE system! Limiting factor! Increase limiting factor  better growth

Limiting factor! Optimize: always look at the WHOLE system! Further increase of oxygen: little effect

Optimize: always look at the WHOLE system! LB medium („Luria broth“): 5 g/L yeast extract 10 g/L Trypton 0.5 – 10 g/L NaCl TB medium („Terrific broth“): 24 g/L yeast extract 12 g/L Trypton 4 mL Glycerin Phosphate buffer

Optimize shaking cultures: Look at the WHOLE system! Shake flask = black box Limiting factor = ??? Optimization: usually try and error Increasing oxygen alone may not be enough Enriched culture medium may be required

Summary: Thomson flasks: - double volume AND - better culture results Multitron Pro can handle the load! Cheaper shakers do not reach the shaking speed Argument for Infors shakers. Process optimization: Always look at the whole system Use enriched medium 26.11.2018

Thank you! 26.11.2018