1. Case study 2

2. Session outline  A sequential modular approach to solve for a moderate complex flowsheet  Some common unit operations in biochemical industries: Fermentor Disk-stack centrifugation Diafiltration Chromatography

3. Sequential modular approach Part 1 Part-2 Purification Section 1 Part 3-Purification Section 2

4. Description Water, microorganisms, nutrients (glucose) and air are fed into a bioreactor where at 37 °C a fermentation takes place yielding an enzyme and impurities. Biomass is separated in a disk-stack centrifuge and the liquid is stored in tank. It is then processed in a diafilter where the remaining biomass is removed (when a small loss of product). It is stored again and then loaded onto a PBA chromatography column where the enzyme binds and is eluted using WFI/NaCl mixture.

5. Fermentation Section  Mode of operation: batch processing  Component registration: Glucose Biomass CO 2 WFI (water for injection) Enzyme (new-water as reference comp.) Impurities ( new-water as reference comp.)

6. Process flowsheeting  Fermentation process Equipment: Vessel Procedure/ In a Fermentor Description: Transformation of raw material into enzyme & impurities  Centrifugation Equipment: Centrifugation/ Disk-Stack Description: separation of biomass  Product storage 1 Equipment: Storage/ Bulk/ Batch/ in a Blending Tank) Rename as: Storage 1 (Right click equipment/ Edit labels) Description: temporary product storage

7. Product initialisation  Fermentation  Centifugation  Storage 1

8. Fermentation  CHARGE-1: Charge 10000 L of water @ 100 L/min  CHARGE-2: Charge 1000kg glucose @ 40kg/min  Heat-1: Final temperature: 37 °C Efficiency: 90% Duration: calculated based on constant heating rate 0.5 °c/min  Ferment-1 (Stoichiometric): Operation condition:  Final temp @37 °C  Heat transfer agent: Cooling water  Process time: 36 hrs  Fermentor aeration: select air from stock mixture (auto adjust) (continue…)

9. Fermentation  Ferment-1 (stoichiometric) (cont.): Reaction (mass stoichiometry):  100Glucose + 80 O2 55 Water + 2 Enzyme +3 Impurities + 80 CO2 + 40 Biomass  Reaction extent: 98% based on limiting component  Enthalpy: -3700 kcal/kg; ref. comp.: O2; ref. temp:37 °C Emission: 100% for CO2 (select “Perform emission calculation” & “Set By User” for CO2)  TRANSFER-OUT-1: duration same as Centrifugation (use Master-Slave relationship) (note: Leave other values as default)

10. Fermentation

11. FERMENT-1 Final temp: 37 °C Process time: 36 hr Aeration setting: Auto adjust for air (stock mixture) Mass stoichiometry Enthalpy data Reaction extent

12. Centrifugation  Only 1 operation: CENTRIFUGE-1 (default)  Operating condition: Equipment design based on: Solid Removal Duration: 3 hours (Centrifugation time)  Material balance: Component removal %: set by User Solid component removal %: 98% for biomass; 0% for others Solids Concentration in Solid Streams: 500 g/L  Utilities: Exist temp @15 °C (Set by User) Agent: chilled water  Scheduling: Start when Transfer-out of Fermentation (P-1) starts

13. Storage 1  TRANSFER-IN-1: Operating conditions:  Transfer in using: outlet stream from centrifuge  Duration: same as Centrifuge (set by Master-Slave Relationship) Scheduling: Start when Centrifugation (P-2) starts operation  STORAGE: Duration: to be determined Scheduling: start when TRANSFER-IN-1 starts

14. Let’s simulate the flowsheet & solve the error message given (scheduling problem)

15. Purification section 1 Please delete “STORE-1” operation in P-3 & replace it with a “Transfer-Out-1”

16. Process flowsheeting  Diafiltration (DF) Equipment: Filtration/ Diafiltration Description: Removal of all leftover biomass from Storage 1 Remark: Storage (P-3) outlet needs to be deleted before new stream can be connected to the diafilter  Product storage 2 Equipment: Storage/ Bulk/ Batch/ In a Blending Tank Rename as: Storage 2 Description: temporary product stage (Note; Right click on equipment & select “Flip (reverse direction)” to turn the equipment into reverse direction

17. Diafiltration descripition  In diafiltration, water or some other solvent or buffer is added to the retentate to facilitate the removal of membrane-permeating species along with the water (or other solvent) during filtration.  The addition of water (or any other solvent) can be conducted either in batch or continuous mode.  In batch operation, permeable solutes are: Cleared from the retentate by volume reduction (batch concentration); Followed by re-dilution with water ( or other solvent); and Re-concentration in repetitive steps

18. Diafiltration in SuperPro  In the current version of SuperPro Designer, batch concentration can precede and follow a continuous operation (true diafiltration)  Any number of batch concentration stages can be specified for each discontinuous operation.  In general, if the initial solution is dilute, a concentration step (to reduce the volume of the material) usually precedes a continuous diafiltration step. Feed tank Recycle Loop Permeate (Filtrate) Retentate (Concentrate)  If the initial solution concentration is rather high, one usually goes directly to continuous diafiltration

19. Procedure initialisation  Diafiltration  Storage

20. Diafiltration  Operating condition: Rejection coefficient (RC): biomass 100%, impurities 20%, enzyme 5% Max. solid concentration in retentate: 600 g/L Product denaturation (denaturation is due to shear forces during membrane filtration, common in bioprocessing):  Denaturation: 4%  Active product: enzyme  Denaturated product: impurities Duration: 4 hrs (filtration time) Diluant: water (auto adjust) Diafiltration data: 5 (Volume Permeated) Concentration data:  Prefiltration: YES, # of conc. stages: 1, Conc. Factor 5  Postfiltration: NO

21. Diafiltration  Utility: Select “set by User” Exit temperature = 15°C Agent: Glycol Specific power: 0.2 kW/m 2  Scheduling: Start with TRANSFER-OUT of Storage 1  Additional task: Set TRANSFER-OUT-1 of Storage 1 (P-3) to follow the duration of Filtration in Diafilter (P-4) using Master-Slave relationship

22. Storage 2  TRANSFER-IN-1: Transfer in using: outlet stream from DF (P-4) Start (scheduling) and duration (Operating condition: Master-Slave) same as DF  STORAGE: Duration: to be determined Scheduling: start when TRANSFER-IN-1 starts

23. Simulate the flowsheet & solve the scheduling

24. Purification section 2 Again, replace “STORE -1” operation in P-5 with “TRANSFER-OUT-1”

25. Process flowsheeting  PBA Chromatography Equipment: Chromatography/ Adsorption/ PBA Chromatography Description: binds and is elutes the enzyme using a WFI/ NaCl mixture (new mixture to be registered)  Product storage 3 Equipment: Storage/ Batch/ In a Blending Tank Rename as: Storage 3 Description: temporary product storage (Note: Right click on equipment & select “Flip (reverse direction)” to turn the equipment into reverse direction

26. New mixture registration  We need a mixture of “NaCl/WFI(2M)” for this section, but this mixture is not found in the component database of SuperPro (verify this from Stock Mixture database)  2 ways of registering this mixture: Register as NaCl (2M) & replace the water compound in this mixture with WFI Register it from scratch & fill in the physical properties that you have

27. Modify from existing mixture  Path: Task/Edit Stock Mixtures Highlight the water component, delete & replace it with WFI Make sure the mass % is make up into 100%

28. Register from scratch  Path: Task/Edit stock Mixtures Create new mixture Choose this option if you know the density of the mixture Choose this option to modify from an existing mixture (e.g. NaCl mixture)

29. Let’s try it … (Always remember to save your work …)

30. Procedure intialisation  PBA Chromatography  Storage 3

31. General description  PBA column loading (Load): estimate the time for loading a column, track recovery yield, & estimate the number and size of columns required  Column washing (Wash): wash away the undesired impurities that trap in the column

32. General description  Column elution (Elute): A column may be used to bind either:  Product component(s); or  Impurity components For retention of product components, for a component that binds to the resin, its amount in the product stream = (amount in the feed stream) x (binding fraction) x (elution yield) All component present in the feed stream, that do not bind to resin, exit into the waster stream  Colum regeneration (Regenerate): regenerate the resin using a solution

33. PBA Chromatography  LOAD-1: Operating condition:  Loading flowrate: 200 cm/h (linear velocity)  Resin’s primary function: Retain Product (default)  Comp binding & yield (Binding refers to all components that bind to the resin; yield represents the percentage of bound material that ends up in the product stream.): -enzyme 100%, 90% -impurity: 20 %, 30% Scheduling: Starts when Transfer-Out of Storage 2 Additional task for Storage 2:  Make sure that the “Storage-1” operation in Storage 2 is replaced by “TRANSFER-OUT-1”  Set TRANSFER-OUT-1 of Storage 2 to have the same duration as LOAD-1 using Master-Slave Relationship

34. PBA Chromatography WASH-1: Volume per cycle: 2 BV (bed volumes) Wash stream: “Wash” stream which contains WFI (auto adjust) ELUTE-1: Eluant Volume: Total Volume: 8 bed vol. Volume in Product Stream: 2 bed vol Flow rate Options: 200 cm/h (linear velocity) Elution Strategy: Gradient Key comp data: Name: NaCl Initial concentration: 0 mol/L Final concentration: 1 mol/L Eluant A: NaCl/WFI (2M) in stock mixture Eluant B: WFI (auto-adjust) REGENERATE-1: Linear velocity: 300 cm/h Volume per Cycle: 2 BV Wash stream: “Regen” stream with WFI (auto- adjust) Additional task: Delete “Equilibrate” operation in P-6

35. Storage 3  TRANSFER-IN-1: Transfer from: PBA chromatography; using outlet stream from PBA chromatography Start (scheduling) and duration (Operating condition: Master-Slave) same as ELUTE-1 in PBA chromatography (Q: why not the last operation?)  TRANSFER-OUT-1: Transfer to: none; using outlet stream from Storage 3 Scheduling: start when TRANSFER-IN-1 completed

36. Check your simulation results  Check the input to your PBA chromatography  Since we specify comp binding & yield for: Enzyme to be 100%, 90% Impurity: 20%, 30%  The amount of enzyme in the product stream: ___kg  The amount of impurities in the product stream should be: ___kg Please check this out & verify this from your simulation results.