Single use systems

Systematic Evaluation of Single-Use Systems Using Process Simulation Tools – A Case Study Involving MAb Production Victor Papavasileiou is a senior applications engineer with Intelligen Europe, Leiden, Netherlands ([email protected]), Charles Siletti, Ph.D. is the director of scheduling and planning applications ([email protected]) and Demetri Petrides, Ph.D. is the president of Intelligen, Inc., Scotch Plains, NJ, USA, +1 908 654-0088 ([email protected]).

Abstract Single-use systems are an option that has gained popularity in the biopharmaceutical industry in the last few years. The choice of single-use vs. stainless steel depends on a variety of process and other parameters, such as bioreactor scale, product titer, product changeover frequency, etc. Computer-aided process design and simulation tools facilitate analysis and evaluation of process alternatives and assist scientists and engineers in their decision making process. This article describes the steps required to build a comprehensive model in a batch process simulator that accounts for the consumption of single-use systems for buffer preparation and storage. The process is subsequently compared to a more traditional one employing stainless steel tanks for preparation and storage of buffers. The impact of single-use systems on production cost, demand for cleaning materials, demand for consumables, and the cycle time of the process is thoroughly evaluated. The analysis is done for a typical cell culture facility producing therapeutic monoclonal antibodies.

Keywords Single-Use Systems, Disposables, Manufacturing of Biopharmaceuticals, Bioprocess Economics, Bioprocess Simulation.

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Introduction As the number of biopharmaceutical molecules that are entering clinical trials is rising, there is increased demand for technologies that can expedite the commercialization process. Disposables or single-use systems constitute such an enabling technology. They are commonly used for inoculum expansion using wave rocking bioreactors that are available for working volumes of up to 500 L.1 More recently, stir tank disposable bioreactors have become available with working volumes of 1,000 L and even 2,000 L aimed at replacing small to medium scale stainless steel bioreactors.2 Preparation and storage of cell culture media and product purification buffers in disposable bags is another common application.3 Their use greatly reduces the need for piping, CIP & SIP infrastructure, and the consumption of cleaning materials.1 This in turn reduces the requirements for up-front capital investment and speeds up the commercialization process. The above attributes of single-use systems make them particularly attractive to start-up companies that are short in capital and are under pressure to meet development milestones. Single-use systems, however, are not a panacea. They result in increased cost of consumables and their application ceases to be advantageous beyond a certain scale of production. Detecting the turning-point scale is a challenging task that depends on process and other parameters. Process simulation and other modeling tools can play an important role in this task by facilitating the analysis and evaluation of alternatives at various scales. The focus of this article is on the role of such tools in the evaluation of process alternatives and in particular the evaluation and comparison of single-use systems versus the traditional stainless steel approach. The evaluation is done for a typical monoclonal antibody (MAb) facility at the clinical manufacturing scale. Two process alternatives are evaluated in detail. In the first option production buffers and media are prepared and stored in traditional stainless steel tanks; in the second option, buffers and media are prepared and stored in single-use bags. Process simulation tools can assist in the evaluation of process alternatives in all the stages of process development and product commercialization by facilitating the following and other related tasks:4,5,6,7 •

Documentation and process understanding

•

Calculation of material and energy balances

•

Sizing of equipment and utilities

•

Cost-of-goods analysis

•

Process scheduling

•

Cycle time analysis and debottlenecking

•

Resource tracking as a function of time

•

Environmental impact assessment

The cost analysis and resource tracking capabilities of such tools are the features predominantly employed in this case study. Capital and operating costs are used to compare the two alternatives at various production scales. The impact of single-use systems on the demand for cleaning materials, cleaning-in-place (CIP) skids, labor, utilities are also considered.

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Commercially Available Simulation Tools Computer-aided process design and simulation tools have been in use in the chemical and petrochemical industries since the early 1960s. Established simulators for those industries include: Aspen Plus and HYSYS from Aspen Technology, Inc. (Cambridge, MA), ChemCAD from Chemstations, Inc. (Houston, TX), and PRO/II from SimSci-Esscor, Inc. (Lake Forest, CA). The above simulators have been designed to model primarily continuous processes and their transient behavior for process control purposes. Most biopharmaceutical products, however, are manufactured in batch and semi-continuous mode. Such processes are best modeled with batch process simulators that account for time-dependency and sequencing of events. In the mid 1990s, Aspen Technology introduced Batch Plus, a recipe-driven simulator that targeted batch pharmaceutical processes. At around the same time, Intelligen, Inc. (Scotch Plains, NJ) introduced SuperPro Designer. The initial focus of SuperPro Designer was on bioprocessing. Over the years its scope has been extended to include modeling of small molecule API and secondary pharmaceutical manufacturing processes. Discrete-event simulators have also found applications in the pharmaceutical industries, especially in modeling of secondary pharmaceutical manufacturing processes. Established tools of this type include ProModel from ProModel Corporation (Orem, UT), Arena and Witness from Rockwell Automation, Inc. (Milwaukee, WI), and Extend from Imagine That, Inc. (San Jose, CA). The focus of models developed with such tools is usually on the minuteby-minute time-dependency of events and on animation of the process. Material balances, equipment sizing, and cost analysis tasks are usually out of the scope of such models. Some of these tools are quite customizable and third party companies occasionally use them as platforms to create industry-specific modules. For instance, BioPharm Services, Ltd. (Bucks, UK) have created an Extend-based module with emphasis on biopharmaceutical processes. MS Excel from Microsoft is another common platform for creating models for pharmaceutical processes that focus on material balances, equipment sizing, and cost analysis. Some companies have even developed Excel applications that capture the time-dependency of batch processes. This is typically done by writing extensive code (in the form of macros and subroutines) in VBA (Visual Basic for Applications) that comes with Excel. The K-TOPS tool from Biokinetics, Inc. (Philadelphia, PA) belongs to this category.

Building a Model in a Batch Process Simulator SuperPro Designer (Intelligen, Scotch Plains, NJ) will be used to illustrate the modeling and evaluation of the MAb manufacturing process alternatives. The first step is to create a flow diagram of the overall process (Figure 4). The various equipment-shaped icons, called Unit Procedures, represent the processing steps required for making a batch of a certain product. The lines that connect the unit procedures represent material transfers. Batch process simulators usually come with a library of unit procedures. A unit procedure represents the set of activities or operations that are carried out in a piece of equipment during a processing step. The Bioreaction procedure (P-11) of Figure 4 includes the following operations: SIP-1, SET UP, TRANSFER-IN-1, TRANSFER-IN-2, FERMENT-1, TRANSFER-OUT-1 and CIP-1 (Figure 1). The dialog in Figure 1 is used for adding operations to a unit procedure. On the left-hand side of the dialog, the program displays the operations that are available for the unit procedure (in this case a Bioreaction procedure); on the right-hand side, it displays the userselected operations. The combination of unit procedures and operations enables the user to represent and model the various activities of batch processing steps in detail.

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Figure 1. Operations associated with the production bioreaction unit procedure.

For every operation of a unit procedure, the simulator includes a mathematical model that performs material and energy balance calculations and equipment-sizing calculations. If multiple operations within a unit procedure dictate different sizes for the equipment, the program reconciles the different demands and selects an equipment size that is appropriate for all operations. If the equipment size is specified by the user, the simulator checks to make sure that the vessel is not overfilled. In addition, the tool checks to ensure that the vessel contents will not fall below a user-specified minimum volume (e.g., a minimum stir volume) for applicable operations. In terms of cost analysis, simulation tools facilitate estimation of capital as well as operating cost. Some tools are equipped with built-in functions and databases for estimating equipment cost as a function of size, material of construction, operating pressure, and other parameters.8,9 They may also include databases for materials (pure components as well as mixtures), utilities, consumables, and other resources. The size and unit cost of single-use systems is stored in the consumables database. The user associates consumables with a processing step and the tool calculates the number of units and the cost (Figure 2).

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2.Specify properties

3.Assign to equipment 1.Create new consumable in database

Figure 2. Specifying single-use systems.

Since biopharmaceutical processes operate in batch mode, the simulator must also facilitate process scheduling and cycle time analysis. The results of process scheduling are typically visualized with Gantt charts that display equipment occupancy as a function of time (Figure 3).

CIP Skids Reuse of Buffer Prep. tanks

Buffer Prep Tanks

Reuse of Buffer Hold tanks

Buffer Hold Tanks

Bioreactors

Multiple (x4) Production Bioreactors

DSP

Figure 3. The equipment occupancy chart for the stainless steel process.

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Sensitivity and parametric analysis are other common benefits from the use of such tools. Input parameters can be varied manually or automatically and their impact can be evaluated in a matter of minutes. If statistical data are available for certain input parameters, their impact on output (decision) variables can be evaluated using Monte Carlo simulation.10 It should be noted, however, that the GIGO (garbage-in, garbage-out) principle applies to all computer models. If critical assumptions and input data are incorrect, so will be the outcome of the simulation. Consequently, a certain level of model validation is necessary. In its simplest form, a review of the results by an experienced engineer can play the role of validation.

Process Description Figure 4 displays the flow diagram of the overall process (the way it is represented in SuperPro Designer). Please note that, for simplicity, the buffer preparation and holding activities are omitted from Figure 4. Those activities, however, were taken into account in the detailed models that were built for the evaluation of the alternatives. The complete flow diagrams (in PDF format) as well as the detailed SuperPro models can be downloaded from the internet by pointing your browser to www.intelligen.com/literature. The computer models can be opened and studied in detail using the free evaluation version of SuperPro Designer which can be downloaded from the internet (www.intelligen.com).

Upstream Processing The inoculum is initially prepared in 225 mL T-flasks. The material is first moved to roller bottles (2.2 L), then to 20 L and subsequently to 100 L disposable bag (wave rocking) bioreactors. The broth is then moved to a stirred-tank seed bioreactor. The media solution for the seed bioreactor (165 L per batch) is prepared in a tank (MP-101) and then sterilized/fed to the reactor through a 0.2 μm dead-end filter (DE-101). Serum-free, low-protein media powder is dissolved in WFI in a stainless steel tank (MP-103). 1,628 L of diluted medium (3%) is prepared per batch. The solution is sterilized using a 0.2 μm dead-end polishing filter (DE-103). A stirred-tank bioreactor (PBR1) is used to grow the cells, which produce the therapeutic monoclonal antibody (Mab). The production bioreactor operates under a fed batch mode. High media concentrations are inhibitory to the cells so half of the media is added at the start of the process and the rest is fed at a constant rate during fermentation. The concentration of media powder in the initial feed solution is 17 g/L whereas the concentration of the medium added during the fed batch phase is 156 g/L. The fermentation time is 12 days. The volume of broth generated per bioreactor batch is approximately 2,000 L, containing roughly 4 kg of product. The product titer is approximately 2 g/L.

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Monoclonal Antibody Production

ProtA-Equiil. ProtA-Wash

S-001 S-003

Inoculum Prep

S-107

ProtA-Elut.

S-108

Primary Recovery

Protein-A

ProtA-Reg. 1.97 L/batch

0.47 L/batch S-002

P-1 / TFR-101

S-034

S-031 S-004

Roller Bottle

P-14 / V-101

P-16 / DE-108

P-15 / DS-101

Surge Tank

S-008

S-006

S-036

P-17 / V-103

Polishing Fitler

Centrifugation S-033

500.70 L/batch

P-19 / DE-109

P-18 / C-101

Centrifugation Pool Tank

PBA Chromatography

Polishing Fitler

ProtA-Waste

S-038

6080.37 L/batch

S-035

S-039

S-009

S-043

S-109

7.70 L/batch

Chemical Virus Inactivation

S-041

30.72 L/batch

S-007 S-05

S-037

S-032

P-2 / RBR-101

T-Flask

S-102

S-103 S-101

P-3 / BBS-101

P-4 / BBS-102

Bag Bioreactor

Bag Bioreactor

S-110

S-045

S-044 S-010

S-046

P-20 / V-107

P-21 / DF-101

P-22 / V-111

Storage

Diafiltration

Virus Inactivation

P-23 / DE-110 Polishing FIlter

S-042 100.15 L/batch

S-048 100.18 L/batch

S-047 S-011 Vent-3 S-030 HIC-Equil

IEX-Equil

S-013 P-7 / DE-101

Media Prep

Sterile Filtration

HIC-Wash

IEX Chrom

IEX-WFI

196.03 L/batch

P-6 / MP-101

Amm. Sulfate

IEX-Wash

S-015

S-012

HIC Chrom

HIC-El

P-5 / SBR1 Seed Bioreactor

S-014

S-016 IEX-El S-1

IEX-Strip

149.14 L/batch

S-049

HIC-Reg

S-051

IEX-Rinse P-25 / V-109

IEX Chromatography S-025

P-27 / DE-106

S-050 175.55 L/batch

IEX Pool Tank

P-24 / C-102

Dead-End Filtration P-26 / C-103 HIC Chromatography

IEX-Waste

S-052

HIC-Waste S-172

1271.56 L/batch

1643.48 L/batch

Bioreaction Vent-5

Viral Exclusion

Final Filtration

S-024 S-026 S-025b

S-028

P-12 / MP-103

P-13 / DE-103

Media Prep

Sterile Filtration

S-106

S-058

S-057

S-054 S-027 1983.06 L/batch

S-028b

P-28 / V-108

S-053

HIC Pool Tank

P-11 / PBR1

S-024b P-34 / MP-104 Media Prep

S-056 P-30 / V-110

P-29 / DE-105 Viral Exclusion Filtration

Production Bioreactor

S-026b

S-059 99.43 L/batch

P-35 / DE-104

S-055

Sterile Filtration

99.43 L/batch

S-062 S-105

S-104

Storage

P-32 / DE-107

P-33 / DCS-101

Final Polishing Filtration

Freeze in 50L Plastic Bags

Final Product

P-31 / DF-102 Diafiltration

S-060

S-061

S-029 S-027b

Figure 4. The flowsheet of the main MAb process.

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Downstream Processing The generated biomass and other suspended compounds are removed using a disc-stack centrifuge (DS-101). During this step, roughly 2% of the product is lost in the solids waste stream. The bulk of the contaminant proteins are removed using a 62 L protein-A affinity chromatography column (C-101) which operates in 4 cycles. The product yield for this step is 90%. The protein solution is then concentrated 5x and diafiltered 2x (in P-21 / DF-101). The total membrane filtration area for the diafilter is 2.6 m2.The yield on product is 97%. The concentrated protein solution is then chemically treated for 1.5 h with Polysorbate 80 to inactivate viruses (in P-22 / V-111). An ion exchange (IEX) chromatography step follows (P24 \ C-102) with a product yield of 90%. The IEX column has a volume of 28 L and the batch is processed in 3 cycles. Ammonium sulfate is then added to the IEX eluate (in P-25 \ V-109) to increase the ionic strength for the hydrophobic interaction chromatography (HIC) step (P26 \ C-103) that follows. The recovery yield of the HIC step is 90%. The HIC column has a volume of 25 L and it processes a batch in 3 cycles. A viral exclusion step (DE-105) follows. This is a dead-end type of filter with a pore size of 0.02 μm. Finally, the HIC elution buffer is exchanged for the product bulk storage buffer (PBS) and concentrated 1.5-fold in DF-102. The approximately 100 L of final protein solution is stored in a 200 L disposable storage bag (DCS-101). 2.5 kg of purified product are produced per batch. The overall yield of the downstream operations is approximately 63%.

Buffer and Media Preparation Several buffers and media are required at different quantities. These have to be prepared and transferred to the use points in time for processing. Two alternatives for the buffer and media preparation activities are investigated. The first option employs single-use preparation and storage systems. Media are prepared in 200 L single-use bags and transported to the use point. The various buffers are prepared in 500 L and 1,000 L bags (utilizing a skid) and then transferred through sterile filters into 200 L storage bags. The 200 L bags are subsequently moved to the point of use. Specifying singleuse bags in SuperPro Designer is a simple process (Figure 2). The first step involves specification of the type of bag, capacity, purchase and disposal costs and other properties in the consumables database. The bag can then be allocated to a unit procedure that represents buffer preparation or storage. The tool calculates the number of required bags per batch and campaign during simulation. Other consumables, such as chromatography resins and membrane cartridges are specified and calculated in a similar manner. In this case study, the three types of bags used have working volumes of 200 L, 500 L and 1,000 L and their assumed purchase costs are $300, $400 and $570 per item, respectively. Table 1. Media and buffer prep and holding tanks for the stainless steel option Tank Name MP-101 MP-102 MP-103 PV-101 PV-102 PV-103 PV-104 PV-105

Size (L) 200 1900 200 3400 1400 900 300 30

Tank Name HV-101 HV-102 HV-103 HV-104 HV-105 HV-106 HV-107 HV-108

Size (L) 3400 1400 900 300 300 400 30 1100

The second option employs traditional stainless steel tanks for buffer and media preparation and holding. Media are prepared and fed using a single tank (dedicated) per bioreactor. The buffer preparation area includes a number of preparation tanks connected to a group of holding tanks using a set of transfer panels. The holding tanks are in turn connected with the 8

main process using buffer delivery lines. The amount and number of buffers that need to be prepared determines the size and the number of the tanks. Table 1 displays the required tanks and their sizes. Three tanks are required for media preparation (MP-101, 102, and 103); five tanks are required for buffer preparation (PV-101, 102, 103, 104, and 105) and eight tanks are required for buffer holding (HV-101 to HV-108). Since, the stainless steel tanks are reused, cleaning in place (CIP) is essential after every buffer preparation batch.

Results and Discussion The main process parameters are summarized in Table 2. The fermentation time is 12 days; the facility is equipped with 4 production bioreactor resulting in a cycle time of 3.5 days. 80 batches can be executed per year (20 per bioreactor). The product titer in the production bioreactors is 2 g/L. With a broth volume of approximately 2,000 L and a downstream yield of 62.5%, the amount of purified MAb produced per batch is 2.5 kg. Table 2. Summary of the main process parameters

Fermentation Time Production Bioreactors Purification Trains Process Cycle Time Batches Per year Product Titer Bioreactor Broth Volume Recovery Yield Batch Throughput Annual Throughput

12 days 4 1 3.5 days 20/bioreactor 2 g/L 2,000 L 64,40% 2.5 kg 202.5 kg

Figure 3 displays the equipment occupancy chart (also known as Gantt chart) for thirteen consecutive batches of the 2,000 L stainless steel process option. The activities (unit procedures) of each batch are displayed with a unique color. The various equipment groups are displayed on the chart. The occupancy of CIP skids is represented by the top six lines. The next two equipment groups correspond to the occupancy of the buffer preparation and holding tanks, respectively. Most of these tanks are reused within a batch. Reuse of a tank (e.g., HV-101) within a batch is represented by multiple rectangles of the same color (one rectangle for each unit procedure utilizing that equipment). Reuse of tanks (for preparing and holding different buffers within a batch) reduces the number of vessels and consequently the capital investment for a new facility. However, it also increases their occupancy and cycle times making them likely scheduling bottlenecks. The next equipment group corresponds to the three seed (SBR1a, b, and c) and the four production bioreactors (PBR1a, b, c, and d). Both the seed and the production bioreactors operate in staggered mode (out of phase) in order to reduce the cycle time of the overall process to 3.5 days. A single downstream line (DSP group) handles all purification batches. The equipment occupancy chart enables users to visualize the utilization of equipment and readily identify equipment scheduling bottlenecks that determine the cycle time of the overall process. The production bioreactors (PBR1a, b, c, and d) have the longest cycle time and constitute the scheduling (or cycle time) bottlenecks for the base case. However, if the number of production bioreactors is increased, then, the bottleneck will shift to the buffer preparation and holding tanks that have the next highest utilization. Scheduling bottlenecks linked to buffer preparation equipment is not a consideration for the disposables option unless the buffer preparation bag skids are reused. 9

In general, media and buffer preparation cycle times for the disposables option are shorter than the stainless steel case because disposable bags do not require SIP or CIP steps. The reduced need for cleaning also has a positive impact on the required number of CIP skids and the volume of cleaning materials. Figure 5 displays the required number of CIP skids (as a function of time) for the stainless steel and the disposables options, respectively. Six CIP skids are required in the first case while this number is halved for the single-use option, which in turn reduces the capital cost of the single-use option. Table 3 provides information on the demand for cleaning materials for the two cases. The use of disposables reduces the volume of cleaning materials by more than 50%.

Figure 5. CIP skid requirements (peaks) for the stainless steel (a) and single-use (b) options.

The two options were also compared from an economic perspective Table 4 and Figure 6 display the breakdown of operating costs for both options and their respective unit production costs. The unit production cost for the disposables option is 24% lower than that of the stainless steel option ($317/g versus $415/g). The facility dependent cost, which mainly accounts for the depreciation and maintenance of the facility, is $27 million per year for the single-use option versus $38 million per year for the stainless steel option. The cost of raw materials, which includes the cost of cleaning materials, is also considerably higher for the stainless steel case. On the other hand, the consumables cost is higher in the case of disposables ($8 million per year versus $5 million per year). Table 3. Demand for WFI, cleaning materials, and clean steam

WFI consumption (L/batch) Caustic solution consumption (L/batch) Acid solution consumption (L/batch) Clean Steam Consumption (kg/batch)

Stainles Steel

Single-Use

Reduction

46,000

16,000

65.2%

11,000

3,500

68.2%

14,000

5,000

64.3%

2,600

1,250

51.9%

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Table 4. Cost-of-goods comparison between the two alternatives

Stainless Steel Cost Item

$

Raw Materials Labor-Dependent Facility-Dependent Other Consumables TOTAL Production Cost ($/g)

Single-Use %

5,870,000 22,628,000 38,093,000 11,314,000 4,946,000 82,937,000 415

7.08 27.28 45.93 13.74 5.96 100

$

%

3,532,000 21,825,000 27,048,000 10,912,000 7,966,000 71,341,000 317

4.95 30.59 37.91 15.38 11.17 100

COG's comparison

100% 90% 80% 70%

Consumables

60%

Other

50%

Facility Dependent

40%

Labor

30%

Raw Material

20% 10% 0% Stainless Steel Process

Single Use Process

Figure 6. Cost-of-goods breakdown for the stainless steel and single-use options.

For the base case comparison it was assumed that both plants manufacture the same product throughout the year (80 batches per year). If frequent product changeovers are required, which is common for clinical manufacturing facilities, then the number of batches per year will go down and the facility dependent cost (per unit of product) will increase. And since single-use systems facilitate product changeovers (due to reduced validation), the advantage of single-use systems will be more profound under those conditions. For the 2,000 L production bioreactor scale, the disposables option is clearly the preferred alternative. The advantage of the disposables option gradually diminishes as the scale is increased (Figure 7). For the scale of 8,000 L the options are roughly equivalent from a costof-goods point of view. The analysis reveals that the single-use systems option for buffer preparation and holding is clearly more economical at smaller scales (under 8,000 L of production bioreactor scale). The main reason is the significantly lower facility-dependent and material costs. The facility-dependent cost is lower in the case of disposables due to reduced requirement for stainless steel vessels, CIP skids, piping infrastructure, and utility systems. The cost of materials is lower due to reduced demand for cleaning materials.

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Production Cost vs Scale 900

Production Costs ($/g)

800

Base Case

700 600 500

Stainless Steel Process

400

Single Use Process

300 200 100 0 1000

2000

4000

8000

Bioreactor Capacity (L)

Figure 7. Unit production cost of the two options at different scales.

For scales larger than 8,000 L (of production bioreactor working volume), the stainless steel option starts becoming more attractive. Firstly, the number of buffer bags that need to be prepared and transported to the point of use becomes impractically high (Figure 8). The volume of the hold bags is limited to 200 L due to the fact that they have to be manually transported on a cart to the point of consumption. In addition, multiple buffer preparation skids are required to avoid bottlenecks associated with preparation of buffers which in turn affects the capital investment. Labor costs are also considerably increased since more operators are required for preparing and transporting the bags; labor demand for the stainless steel case does not change much with scale. It is important to note, however, that the 8,000 L scale is not a universal turning point for MAb processes. Increased product titers will most likely result in lower turning points since they are equivalent to higher batch throughputs.

200L Single Use Bags (Items/batch)

200L Bags vs Production scale 250 230 210 190 170 150 130 110 90 70 50 2000

4000

8000

Bioreactor Capacity (L)

Figure 8. Number of 200 L single-use bags required per batch as a function of production scale.

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Summary This article demonstrated how to employ process simulation tools in order to assist in the evaluation of process alternatives via an illustrative case study. Two options for buffer preparation and holding activities of a typical MAb process were analyzed from an economic perspective. The first was employing single-use buffer preparation and storage bags whereas the second the more traditional stainless steel preparation and holding tanks. The single use system proved to be more advantageous for smaller scales whereas the stainless steel option becomes more economical as the production scale increases. The results of the evaluation are specific to the MAb process analyzed. Additional process options and alternative technologies can be readily evaluated and compared using such tools.

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