Scale up/scale down strategies and devices 16/11/2016

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2 Scale up/scale down strategies and devices 16/11/2016 Valentina Mangiafridda MEng Gas Lab and Fermentation Manager 2016 Centre for Process Innovation Limited. All Rights Reserved.

3 CPI mission We use applied knowledge in science and engineering combined with state of the art facilities to enable our clients to develop, prove, prototype and scale-up the next generation of products and processes. Industrial Biotechnology and Biorefining How is it possible to give solid and reliable answers to scale up challenges at early research stage?

4 The instrument we need should help us... to forecast process results in Demo scale... to mimic and predict the client manufacturing scale processes... to easily increase the consistency in process performance through process characterisation... to evaluate the scalability of our process in development Challenges covered with Scale Down Model (SDM) Approach 750L 5/10/20L 1L 1mL

5 Scale Down Model Design Bioprocesses performances are multiphase systems: gas-liquid, or three-phase; Mass transfer between the phases controls process kinetics.

6 Scale Down Model Design The approach is based on the mass transfer uniformity among the scales Its aim is to have the same k L a during the process to transfer This must accurately reproduce the event of a process (ph drifting, media sterilisation peculiarities, parameters control inefficiencies, etc..)

7 Scale Down Model Design The data collected and analyses are: INPUT Bioreactor design & Mass Transfer performances Process parameters (setpoint & controls) po 2, Air Flow, stirrer speed, ph, Head pressure, Temperature,... OUTPUT Fermentation batch Dynamic behaviour of the process Growth curves Output Product, Metabolites, Impurities (with a Kinetic study during the process) Bibl. Villadsen at al., Bioreaction Engineering Principles, 2011

8 Scale Down Model Design Impeller performances with gas dispersion retrofit comparison Bakker A Fermenter Specific Modeling Issues

9 SDM step-by-step A rigorous approach need to be followed to de-risk a bioprocess technical transfer Collect the Bioreactor design and K L adata Analysis on the geometrical parameters Comparison of k L amethod determination and values Bioreactor set up and redesign Mixing Characterization Culture conditions to apply in the new bioreactor

10 Example rprotein production in E. coli Scale (L) Gas velocity (m/s) Maximum impeller speed (RPM) Harvest OD 600 Protein Yield (g/l) k L a (s -1 ) x ± x ± x ± Scale (L) The SDM aim is to have the same k L a across scales Gas velocity (m/s) Maximum impeller speed (RPM) Harvest OD 600 Protein Yield (g/l) x ± x ± x ± k L a (s -1 ) P. Farrell et al. / Vaccine 30 (2012)

11 CPI case study (750L 20L) GC of 750L vs 20L SDM 750L batch fermentation to replicate on the 20L SDM 750L 20L

12 How measure the k L a? The most used method is the Dynamic Gassing-out method Slope: k L a dc dt AL = k L * ( CAL ) qo X a C AL 2 Process values to to apply during the ABSORPTION - Stirrer Speed - Inlet gasses flow - Temperature - Pressure k L a= f(stirrer speed, inlet gasses flow)

13 Question Tech- Transfer based Project for the scale-up of aerobic process for enzyme production at 750L. The client performed in 1L scale the process at maximum stirrer speed and Air flow with remarkable results in terms of Oxygen supply during the C-source feeding The K L a in these condition is 240 h -1 The maximum K L a achievable in 750L is 175 h -1. Which Issue can we forecast? The direct scale up will show a severe Oxygen limitation, with production of side-products Riduction of productivity in large scale Run Lost!!

14 Question Aim: To define scalable Fed-batch aerobic process for enzyme production at 750L. The maximum K L a achievable in 750L is 175 h -1. Study will be focus on Glucose profile and the effect to DOT control at defined mass transfer in 5L bioreactor. The mass transfer condition will be scaled down to 5L Which conditions will give a K L a of 175 h -1?

15 5L Mixing Characterisation Results Internal CPI data K L a in 5L bar 37degC Gas-liquid flow regimes KLa (h-1) Air Flow (vvm) Stirrer Speed (rpm) Bakker A Fermenter Specific Modeling Issues

16 Time for your calculations KLa dep. Stirrer Speed KLa dep. Air Flow KLa (h-1) vvm 0.5 vvm 1 vvm KLa (h-1) rpm 400 rpm 800 rpm Stirrer Speed (rpm) Air Flow (vvm)

17 Time for your calculations The maximum Air flow in 750L is 1vvm KLa dep. Stirrer Speed y = 0.323x R² = h -1 can be reached with 556rpm KLa (h-1) vvm 0.5 vvm 1 vvm Stirrer Speed (rpm)

18 Solution The Scale Down Model limit the stirrer speed and air flow cascade ranges to 556rpm and 1vvm The C-source feeding will be reduced once reached the DOT set point The C-source feeding will be scaled up as compromise for a continuous enzyme production in presence of sufficient Oxygen

19 Other features. SDM information to be integrated with the CFD analysis, in order to fully understand the overall mass transfer distribution in the bioreactor. Bakker A Fermenter Specific Modeling Issues

20 Other features. 1L 1mL In SDM scale the variability of your model & process can be determinate. Process optimisation and Process Characterisation can be done with DoE studies in that scale.

21 Summary Bioprocesses are prone to mixing related performance problems: Gas dispersions can never be homogenous. Mass transfer will limit productivity or affect process control. A rigours model needs to be establish to forecast common issues typical of large scale bioreactor Scale down model (SDM) k L a based can help to to evaluate the scalability of our process in development to forecast process results in Demo scale Coupled with CFD and DoE Model, represent the next step for a completer understanding of Mid-stream steps in bioprocesses

22 Demonstration in the lab Validate results of the previous question: 556rpm and 1vvm will give a k L a of 175h -1? K L a

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