1. ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011) Modeling & Simulation of Bioprocess: ‘BIOCHEMICAL CASE STUDY’ By; Mrs Hafiza Binti Shukor

2. Student should be able to;  Develop chromatographic process to achieve the desired final product purity, combination with membrane filtration to exchange buffers and concentrate the dilute product solutions.  Create the process and uncertainty problem that might happen during the operations. ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

3. OUTLINE FOR BIOCHEMICAL CASE STUDY  A sequential modular approach to solve for a moderate complex flowsheet  Some common unit operations in biochemical industries:  Fermentor  Disk-stack centrifugation  Diafiltration  Chromatography ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

4. Sequential modular approach Part 1 Fermentation Section Part 2 Purification Section 1 Part 3 Purification Section 2 ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

5. PROCESS DESCRIPTION separated in a disk-stack centrifuge and the liquid is stored in tank. (storage 1) Part 1 Fermentation Section Fermentation Unit Bioreactor Feed Water Microorganism Nutrients (glucose) Condition37 °C Output (yield)Enzyme + Impurities Biomass ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

6. Part 2 Purification Section1 Part 3 Purification Section 2 DiafiltrationRemove remaining Biomass Blending/ StorageStore product (small loss of product) PBA Chromatography Column Bind the enzyme And eluted by using WFI/NaCl mixture Blending/ StorageStore product ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

7.  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.) Part 1 Fermentation Section ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

8. Process Flowsheeting for Fermentation Section UNIT PROCEDURE EQUIPMENTDESCRIPTION FermentationVessel Procedure/ In a Fermentor transformation of raw material into enzyme & impurities CentrifugationCentrifugation/ Disk- Stack separation of biomass Storage 1 Storage/ Bulk/ Batch/ in a Blending Tank) Rename as: Storage 1 (Right click equipment/ Edit labels) temporary product storage ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

9. PRODUCT INITIALISATION FOR FERMENTATION SECTION  Fermentation  Centifugation  Storage 1 ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

10. FERMENTATION Initialising CHARGE operation (right click on unit procedure (Fermentation)then click add /remove operations.. Add Charge -1, Charge-2, Heat-1, Ferment-1, Transfer-Out-1. ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

11. CHARGE OPERATIONDATA CHARGE-1Charge 10000 L of water @ 100 L/min CHARGE-2Charge 1000kg glucose @ 40kg/min HEAT-1 Final temp : 37 °C Efficiency: 90% Duration: calculated based on constant heating rate 0.5 °c/min TRANSFER-OUT-1 duration same as Centrifugation (use Master-Slave relationship) Continue… ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

12. Continue… CHARGE OPERATION DATA 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) Reaction (mass stoichiometry); 100Glucose + 80 O 2 55 Water + 2 Enzyme + 3 Impurities + 80 CO 2 + 40 Biomass Reaction extent: 98% based on limiting component Enthalpy: -3700 kcal/kg; ref. comp.: O 2 ; ref. temp:37 °C Emission: 100% for CO2 (select “Perform emission calculation” & “Set By User” for CO2) Note: Leave other values as DEFAULT ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

13. FERMENT-1 Final temp: 37 °C Process time: 36 hr Aeration setting: Auto adjust for air (stock mixture) Mass stoichiometry Enthalpy data Reaction extent ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

14. CENTRIFUGATION UNIT PROCEDURE OPERATING CONDITION MATERIAL BALANCE UTILITIESSCHEDULING CENTRIFUGE-1 (default) Equipment design based on: Solid Removal Duration: 3 hr (Centrifugation time) Component removal %: set by User Solid component removal %: 98% for biomass; 0% for others Solids Concentration in Solid Streams: 500 g/L Exist temp: 15 °C (Set by User) Agent: chilled water Start when Transfer-out of Fermentation (P-1) starts ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

15. STORAGE 1 CHARGE OPERATION DATA 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 1Duration: to be determined Scheduling: start when TRANSFER-IN-1 starts ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

16. Let’s simulate the flowsheet & solve the error message given (scheduling problem) ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

17. PURIFICATION SECTION 1 Please delete “STORE-1” operation in P-3 & replace it with a “Transfer-Out-1” Right click on storage; then click on add/remove operations/delete storage/add TRANSFER-OUT-1 ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

18. Process Flowsheeting for Purification Section 1 UNIT PROCEDURE EQUIPMENTDESCRIPTION Diafiltration (DF)Filtration/ Diafiltration 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 2Storage/ Bulk/ Batch/ In a Blending Tank Rename as: Storage 2 Temporary product stage (Note; Right click on equipment & select “Flip (reverse direction)” to turn the equipment into reverse direction ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

19.  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. PROCESS DESCRIPTION ; Diafiltration  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 Feed tank recycle ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

20. 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.  If the initial solution concentration is rather high, one usually goes directly to continuous diafiltration Feed tank Recycle Loop Permeate (Filtrate) Retentate (Concentrate) ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

21. PRODUCT INITIALISATION FOR PURIFICATION SECTION-1  Diafiltration  Storage 2 ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

22. DIAFILTRATION UNIT PROCEDURE OPERATING CONDITIONUTILITIESSCHEDULING DIAFILTER-1 (diafiltration) 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 Select “set by User” Exit temperature = 15°C Agent: Glycol Specific power: 0.2 kW/m 2 Start with TRANSFER-OUT of Storage 1 ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

23. Continue….. Additional task: Set TRANSFER-OUT-1 of Storage1 (P-3) to follow the duration of Filtration inDiafilter (P-4) using Master-Slave relationship ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

24. STORAGE 2 UNIT PROCEDURE OPERATING CONDITIONSCHEDULING TRANSFER-IN-1Transfer in using: outlet stream from DF (P-4) Start (scheduling) and duration (Operating condition: Master- Slave) same as DF STORAGE 2Duration: to be determinedScheduling: start when TRANSFER- IN-1 starts ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

25. Simulate the Flowsheet & Solve The Scheduling Error ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

26. PURIFICATION SECTION 2 Again, replace “STORE -1” operation in P-5 with “TRANSFER-OUT-1” ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

27. (Note: Right click on equipment & select “Flip (reverse direction)” to turn the equipment into reverse direction Process Flowsheeting for Purification Section 2 UNIT PROCEDURE EQUIPMENTDESCRIPTION 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 3Storage/ Batch/ In a Blending Tank Rename as: Storage 3 temporary product storage ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

28. 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: A) MODIFY FROM EXISTING MIXTURE ‘Register as NaCl (2M) & replace the water compound in this mixture with WFI’ B) REGISTER FROM SCRATCH Register it from scratch & fill in the physical properties that you have ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

29. A)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% ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

30. B)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) ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

31. Let’s try it … (Always remember to save your work …) ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

32. PRODUCT INITIALISATION FOR PURIFICATION SECTION 2  PBA Chromatography  Storage 3 ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

33. GENERAL DESCRIPTION : PBA CHROMATOGRAPHY 4 Types of PBA Chromatography Column; 1)PBA Column Loading (Load) 2)Column Washing (Wash) 3)Column Elution (Elute) 4)Column Regeneration (Regenerate) ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

34. COLUMNDESCRIPTION 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 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 ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

35. CHARGE OPERATION OPERATING CONDITION SCHEDULINGADDITIONAL TASK LOAD-1Loading flowrate: 200 cm/h (linear velocity) Resin’s primary function: Retain Product (default) Comp binding & yield; -enzyme 100%, 90% -impurity: 20 %, 30% Info; Binding refers to all components that bind to the resin; Yield represents the percentage of bound material that ends up in the product stream. Starts when Transfer-Out of Storage 2 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 ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

36. CHARGE OPERATION OPERATING CONDITION ADDITIONAL TASK WASH-1 Volume per cycle: 2 BV (bed volumes) Wash stream: “Wash” stream which contains WFI (auto adjust) Additional task: Delete “Equilibrate” operation in P-6 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 ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

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38. STORAGE 3 CHARGE OPERATION DATASCHEDULING 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 Start when TRANSFER-IN-1 completed ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

39. 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. ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)

40. Biochemical Case Study REPORT Prepare a detail report of the BIOCHEMICAL CASE STUDY and attached together your simulation result (Gantt Chart) This INDIVIDUAL report is COMPULSORY to submit on Friday (24 Sept. 2010) ERT 315/4 SIMULATION OF BIOPROCESS SEM 1 (2010/2011)