-Galactosidase Production

--------------------------------------------------------------------------------------------------------This example deals with -galactosidase (b-Gal), an intracellular enzyme produced by Escherichia coli. This enzyme is normally produced by E. coli up to 1-2% of total cell protein under conditions of induction of the lac operon. Using genetic engineering, the level can go up to 20-25% of total protein. In this example, an easily attainable level of 10% is assumed. b-Gal has found limited industrial applications until now. It is mainly used in the utilization of cheese whey. More specifically, immobilized reactors with b-Gal have been developed to convert lactose found in cheese whey to glucose and galactose, yielding a sweetened product which can be used as an additive to ice cream, egg-nog, yogurt, and other dairy products. Another application of b-Gal is in the treatment of milk products. A significant number of people are lactose intolerant and cannot digest milk or milk products. Production of lactose-free milk products (using b-Gal reactors) allows those people to digest them. This example analyzes a plant that produces 18,515 kg of b-Gal per year in 216 batches. Three files have been included with this example: BGal-a: This file represents the process at an early stage of development. All equipment is in Design Mode, meaning that all equipment volumes and rates are calculated as opposed to being specified by the user. BGal-b: This file shows the process after equipment sizes have been specified for key pieces of equipment. The BGal-b file was used to produce the tables and graphs in the rest of this document. BGal-c: This file is the same as BGal-b, except that final product formulation and packaging unit procedures have been added.

PROCESS DESCRIPTION (file BGal-b)

Flowsheet Sections A flowsheet section is a group of unit procedures that have something in common. The b-Gal flowsheet has been divided into three sections: 1) Fermentation, 2) Primary Recovery, and 3) Purification (see below). All the unit procedure icons of the Fermentation section are displayed in black, while the icons of the Primary Recovery and Purification sections are displayed in blue and green, respectively. For information on how to specify flowsheet sections and edit their properties, please see Chapter 7.1 of the manual. Fermentation Section Fermentation media is prepared in a stainless steel tank (V-101) and sterilized in a continuous sterilizer (ST-101). A compressor (G-101) and an absolute air filter (AF-101) provide sterile air to the fermentor (FR-101). Gaseous ammonia is used as nitrogen source. Primary Recovery Section The first step of the primary recovery section is cell harvesting to reduce the volume of the broth and remove extracellular impurities; it is carried out by a membrane microfilter (MF-101); the broth is concentrated two-fold. Since b-Gal is an intracellular product, the next step is cell disruption, performed in a high-pressure homogenizer (HG-101). After homogenization, a diskstack centrifuge (DS-101) is used to remove most of the cell debris particles, followed by a dead-end polishing filter (DE-101), that removes the remaining cell debris particles. Finally, the resulting protein solution is concentrated two-fold by an ultrafilter (UF-101). Purification Section Next the product stream is purified by an ion exchange chromatography column (C-101). Then it is concentrated three-fold by a second ultrafiltration unit (UF-102) and polished by a gel filtration unit (C-

102). A diafiltration unit (DF-101) exchanges the gel filtration buffer, and the protein solution is lyophilized in a freeze dryer (FDR-101).

OVERALL MATERIAL BALANCES

The table below provides information on raw material requirements for the entire flowsheet. The quantities are displayed in kg/year, kg/batch, and kg/kg MP (main product). Around 86 kg of purified bGal is produced per batch. Note the large amounts of buffers and WFI that are required per batch.

BULK MATERIALS (ENTIRE PROCESS)

Material
Process Water Glucose Salts H3PO4 (5% w/w) NaOH (0.5 M) WFI Air Ammonia NaCl (0.1 M) NaCI (0.5 M) Tris Buffer TOTAL

kg/yr
16,723,431 1,326,888 221,616 2,609,488 22,275,903 48,102,495 9,486,829 85,320 29,774,876 2,524,995 35,299,940 168,431,781

kg/batch
77,423.292 6,143.000 1,026.000 12,080.962 103,129.182 222,696.737 43,920.505 395.000 137,846.648 11,689.791 163,425.647 779,776.763

kg/kg MP
912.385 72.391 12.091 142.367 1,215.313 2,624.343 517.576 4.655 1,624.437 137.757 1,925.870 9,189.185

The above table was extracted from the RTF version of the Materials & Stream Report.

RESOURCE TRACKING
In addition to estimating the total raw material amount consumed, the user has the option to view charts that display material consumption over time. The following chart displays WFI consumption over 5 consecutive batches. To generate this chart, select Charts \ Materials \ Entering \ Multiple Batches from the main menu bar of SuperPro and then select the material of interest in the dialog that appears (WFI in this case). Similar charts can be generated for other resources such as Labor and Utilities. The red and blue lines correspond to the LHS axis and represent instantaneous and averaged (for 12-h intervals) WFI demand, respectively. The green line corresponds to the RHS axis and represents cumulative demand (for 12-h intervals). This chart provides useful information for sizing WFI systems. Specifically, the tallest red peak (highest instantaneous demand) is useful information for sizing the pipe diameter of the circulation loop and its pumping capacity since the loop and the pump must be able to accommodate the highest instantaneous demand. The tallest green peak provides useful information for sizing the surge tank of the WFI system. It corresponds to the working volume of the surge tank if a 12-h buffer capacity is required (i.e., a 12-h supply even if the still is not operational during that period). The blue line for the green peak interval (which is also the blue peak) provides useful information for sizing the still. The averaging interval can be adjusted by the user. The larger the averaging interval, the greater the size of the surge tank and the smaller the size of the still. In other words, there is a trade off between still size and tank size in the sizing of WFI systems.

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SuperPro also can calculate and display inventory information for material resources and utilities. Suppose there is a 100,000 kg WFI storage tank. Suppose further that the WFI still has a rate of 20,000 kg/h and it is turned on when the level in the tank drops below 35% and off when it reaches 100%. To visualize the liquid level in the tank and the operation of the still, do the following. Select the menu item Chart \ Materials \ Entering, Inventory \ Multiple Batches . Select WFI and then click on the Supply Info button to specify the size of the tank, the rate of the still, the initial contents, and the on/off criteria (the values provided above). Then click OK and on the next dialog click OK again. That will bring up the graph of the figure that follows. The green line (that corresponds to the y-axis on the RHS) represents the WFI level in the tank. The blue line (that corresponds to the y-axis on the LHS) represents the operation of the still.

PROCESS SCHEDULING
The figure below is an Equipment Occupancy chart generated by SuperPro Designer. To generate it select the menu item Chart \ Equipment Occupancy \ Multiple Batches. It shows the execution of the various processing steps as a function of time for five consecutive batches. The overall batch time is approximately 159 h. The recipe cycle time (time between consecutive batches) is 36 h. The minimum recipe cycle time (minimum time interval between consecutive batches) is 33.95 h and is determined by P-16 (in V-105), which is the cycle time bottleneck. The number of batches per year is 216. Parameters related to cycle times and number of batches per year can be specified by selecting Tasks \ Recipe Scheduling Information . The same dialog is accessed through the Equipment Occupancy chart by right-clicking on the white area of the chart and selecting Edit Recipe Scheduling Info . Please note that step P-13 utilizes two tanks (V-104 and V-104b) that operate out of phase (in staggered mode) to reduce the recipe cycle time. Staggered equipment is specified through the Stagger Mode box of the Equipment Data dialog of a procedure (right click on a procedure icon and select Equipment Data). Tank V-104 is the only one displayed on the flow diagram, to distinguish it from non staggered equipment, the following icon appears on the bottom LHS of the procedure icon ( ). Steps P-16 and P19 utilize two tanks operating is stagger mode as well. CIP skids are shown at the top of the chart. Two skids are shared by the equipment in this process. Some CIP operations (see P-13 in V-104) have been slightly delayed to resolve CIP scheduling conflicts.

SuperPro also generates Operations and Equipment Gantt charts for single and multiple batches. The figure that follows displays a portion of the operations Gantt chart for a single batch of this example process. The left view (spreadsheet view) displays the name, duration, start time, and end time for each activity (e.g. each operation, unit procedure, cycle, batch, etc). You can use the left view to expand or collapse the activity summaries by clicking on the + or signs in the boxes to the left of the activity names. The right view (chart view) displays a bar for each activity. To edit the scheduling data (or any other data affecting an activity), simply right-click on a bar and a relevant command menu will appear. Selecting the uppermost entry on this menu will bring up a dialog that allows you to edit the information associated with that particular activity . In fact, anything you can accomplish with the other scheduling interfaces, you can also accomplish from the Gantt chart interface. Furthermore, you can redo the M&E balances and have the Gantt chart updated to reflect the new (calculated) scheduling settings for the recipe by clicking on the Update Chart entry in the main menu of the interface (or the equivalent button on the toolbar). You may also export the Gantt chart to MS Project through the File menu of the chart or the File menu of the application.

COST ANALYSIS AND ECONOMIC EVALUATION

The key results of cost analysis for a plant producing 18,515 kg of b-Gal per year follow. This analysis assumes that a new facility will be built for this process, and the project lifetime is 15 years.

EXECUTIVE SUMMARY (2010 prices)

Total Capital Investment Capital Investment Charged to This Project Operating Cost Cost Basis Annual Rate Unit Production Cost Total Revenues Gross Margin Return On Investment Payback Time IRR (After Taxes) NPV (at 7.0% Interest) MP = Flow of Component B-Gal in Stream Product The above table was copied from the Economic Evaluation Report generated by SuperPro Designer. This table gives an overall summary of the economics of the process. The information above (as well as additional information) is also displayed on the Executive Summary dialog ( View \ Executive Summary). Much more detailed information is available in additional tables of the Economic Evaluation Report and the Itemized Cost Report. One such table is the Annual Operating Cost summary shown below. This breaks down the costs into general categories. From this table, it is clear that consumables are the most important cost. The cost of raw materials is in the second position and facility-dependent costs in the third. Facility-dependent costs account for depreciation, maintenance, local taxes, etc. 141,938,000 141,938,000 114,614,000 18,514.49 6,190.49 183,293,000 37.47 37.49 2.67 28.36 237,753,000 $ $ $/yr kg MP/yr $/kg MP $/yr % % years % $

ANNUAL OPERATING COST (2010 prices) - PROCESS SUMMARY

Cost Item
Raw Materials Labor-Dependent Facility-Dependent Laboratory/QC/QA Consumables Waste Treatment/Disposal Utilities Transportation Miscellaneous Advertising/Selling

$
19,268,000 10,685,000 23,588,000 1,490,000 58,729,000 446,000 406,000 0 0 0

%
16.81 9.32 20.58 1.30 51.24 0.39 0.35 0.00 0.00 0.00

Running Royalties Failed Product Disposal TOTAL

0 0 114,614,000

0.00 0.00 100.00

The graphical representation of the above table is shown in the figure below. This chart is also part of the Economic Evaluation report and is generated if the Include Charts flag is turned on in the Report Options dialog. To access the report options select Reports \ Options from the main menu bar of the application.

The table below (also extracted from the Economic Evaluation Report) provides detailed info on the cost of consumables. It is obvious that the chromatography resins are the major contributors of this cost.

VARIOUS CONSUMABLES COST (2010 prices) - PROCESS SUMMARY

Consumable
Dft DEF Cartridge MF Membrane (Biotech) UF Membrane (Biotech) INX Biotech Resin Gel Filtration Resin TOTAL

Units Cost ($)

600.000 600.000 800.000 1,500.000 800.000

Annual Amount
1,080 item 346 346 17,650 38,903 m2 m2 L L

Annual Cost ($)

648,000 207,360 276,480 26,474,955 31,122,577 58,729,371

%
1.10 0.35 0.47 45.08 52.99 100.00

The Itemized Cost Report displays breakdowns of various costs for each section of the process. For instance, the table below provides information on the contribution of the Fermentation, Primary Recovery, and Purification sections to the process costs. The Purification section contributes 84.02% of the total operating cost. Overall, around 91% of the total operating cost is associated with the Primary Recovery and Purification sections. This is quite common for high-value biological products produced using E.coli.

SUMMARY PER SECTION

Section
Fermentation Section Primary Recovery Section Purification Section TOTAL

$/kg MP
574.944 506.738 5,108.811 6,190.493

$/batch
49,281 43,435 437,903 530,620

$/year
10,644,790 9,382,007 94,587,059 114,613,856

%
9.29 8.19 82.53 100.00

THROUGHPUT ANALYSIS
The Throughput Analysis Report (THR) and the corresponding charts provide information on equipment capacity and time utilization and identify the likely throughput bottleneck. For instance the figure below displays the Utilization chart that is generated by selecting Chart \ Throughput Analysis \ Utilization Factors. The blue color corresponds to size/throughput utilization, the cyan to time utilization, and the magenta to combined utilization. Column C-101 is identified as the most likely throughput bottleneck because it has the highest combined utilization (the product of size and time utilization). Please see the Users Guide and/or consult the Help Facility for more information on throughput analysis and debottlenecking.

STORAGE UNITS
Storage units in SuperPro Designer are employed in order to allow the user to supply or receive materials needed or generated during a production process. Receiving tanks, in particular, may be used to receive the material of output streams as well as waste generated by cleaning-in-place (CIP) operations. Two receiving storage units were added to BGal-b with the following names: Biowaste-Tank and Aqueous-Waste-Tank. The Biowaste-Tank collects waste containing recombinant biomass that needs to be thermally treated (for biomass inactivation) before its release into the wastewater treatment plant. The Aqueous-Waste-Tank collects all other waste. Storage units are not displayed graphically on the flowsheet. Storage units are added to a SuperPro model by selecting Tasks \ Other Resources \ Material Storage Units. Supply and Receiving storage units are displayed on separate tabs. A storage unit can be added by clicking on the magic wand button. Selecting a unit and clicking on the spectacles button brings up a dialog through which the user can view and modify the properties of a storage unit. The properties dialog includes several tabs. The first is the properties tab through which the user may specify the name of the unit, classify the type of waste, and provide other information such as waste treatment cost. The second tab corresponds to the availability limits of the storage unit. The third tab is the inventory data tab which will be discussed in detail below and the final tab is the locations tab which displays the procedures, operations, and streams which utilize the unit. In order to associate a waste stream with a storage unit, right click on the stream to access the context menu and choose the option Assign Receiving Storage Unit . Here the desired storage unit may be selected from the available ones through the drop down menu. In this example, the streams generated during centrifugation and depth filtration named Debris 1 and Debris 2, respectively, were assigned to the Biowaste-Tank. All other waste streams were assigned to the Aqueous-Waste-Tank. Besides output streams, waste generated during CIP operations may also be assigned to the receiving units. To assign CIP generated waste to storage units, open the dialog of a CIP operation and locate the Waste box in the bottom left corner. When a receiving tank is selected, the waste is automatically classified according to the type of waste which has been defined for the unit (e.g. aqueous waste, organic waste, etc.). If the Remove Contents as Waste with Disposed Agent box is checked for a CIP step, any material present in the equipment prior to the CIP operation is removed as CIP waste. For the BGal example, CIP waste generated during biomass containing procedures was assigned to the Biowaste-Tank. This includes the CIP operations of the following procedures: P5 (Fermentation), P7 (Storage), P9 (Homogenization), P10 (Storage), and P11 (Centrifugation). All other CIP waste was assigned to the Aqueous-Waste-Tank. Storage Unit receiving and inventory charts may be generated once the material balances have been solved and provide useful information. Lets first consider the Biowaste-Tank. The receiving chart for this tank can be generated by selecting Charts \ Receiving \ Multiple Batches and selecting the storage unit of interest. The figure below shows the instantaneous deposit rate (magenta lines; corresponding to the y-axis on the LHS)) and the two-day cumulative deposit amounts (green lines; corresponding to the y-axis on the RHS). If two biowaste tanks are available that alternate between collection and treatment every two days, the highest green peak (around 40,000 kg) provides the minimum required size for each.

The figure below displays the instantaneous deposit rate (magenta lines; corresponding to the y-axis on the LHS)) and the 8-h cumulative deposit amounts (green lines; corresponding to the y-axis on the RHS) for the Aqueous-Waste-Tank. This chart indicates that a tank with approximately 160 m 3 of working volume (the tallest green peak) is necessary if an 8-h aqueous waste storage capacity is required.

If the user specifies the size of the Aqueous-Waste-Tank, the rate of the discharge pump, and the discharge criteria, SuperPro can calculate and display the inventory of the tank by selecting Charts \

Receiving Inventory \ Multiple Batches. The values of the above variables can be specified through the Inventory Data tab of the units properties dialog. The figure below displays the inventory for a tank size of 160 m3 and a pump rate of 50,000 kg/h. The discharge pump is turned on when the liquid level reaches 90% of the tanks capacity. The brown line of the chart depicts the inventory and the green line represents the operation of the discharge pump. The properties of storage units also can be accessed through the SUs tab of the Process Explorer pane. The Process Explorer pane is displayed by selecting View \ Process Explorer or by clicking on the Process Explorer button ( ) of the main toolbar.

PRODUCT FORMULATION, PACKAGING, AND DISTRIBUTION

The BGal-c design case file displays a modified version of the base case flowsheet that includes unit procedure models for product formulation, packaging, and distribution. Most of these models are rather simple and their primary objective is to capture the time, materials, and costs associated with such activities. Product formulation and packaging operations often involve formation and use of discrete entities, such as tablets, bottles, boxes, etc. The flow of such entities is represented by discrete streams, which by default are displayed in blue color. For more information on discrete streams and entities, please consult the Help Facility and the Users Guide. To become familiar with the formulation and packaging unit procedure models and the concepts of discrete streams and entities, please open the BGal-c design case and visit the simulation data dialog

windows of these objects. You can do that by right clicking on the various packaging unit procedure icons and their corresponding streams and selecting Operation (or Simulation) Data. Notice the different interface of discrete streams, which display the flow of discrete entities as well as the equivalent bulk flow (based on the bulk ingredients that compose the discrete entities).