Comparison between Batch and Continuous Monoclonal Antibody

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A Comparison Between Batch and Continuous Monoclonal Antibody Production and Economic Analysis Ou Yang, Siddharth Prabhu, and Marianthi G. Ierapetritou Ind. Eng. Chem. Res., Just Accepted Manuscript • DOI: 10.1021/acs.iecr.8b04717 • Publication Date (Web): 30 Jan 2019 Downloaded from http://pubs.acs.org on February 3, 2019

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Industrial & Engineering Chemistry Research

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A Comparison Between Batch and Continuous Monoclonal Antibody Production and Economic Analysis

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Ou Yang, Siddharth Prabhu, Marianthi Ierapetritou*

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Department of Chemical and Biochemical Engineering, Rutgers—The State University of New Jersey, 98 Brett Road, Piscataway, New Jersey 08854-8058, United States

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Corresponding author email address: [email protected]

Abstract

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This work aims to evaluate the benefits of the continuous process in biopharmaceutical manufacturing of monoclonal antibodies (mAbs). An integrated continuous process is designed and built using a process simulator and compared with a fed-batch process production line. The comparisons are based on cost of goods (COG/g) calculation and sensitivity analysis. The fed-batch process results in a total of COG/g of $99/g in mAbs production whereas continuous process accounts for $51/g. Due to the smaller footprint and fewer storage tanks required in the continuous process, the facility cost reduced by 66% compared to the fed-batch. In continuous production there is a better utilization of the primary capture resin, which leads to a reduction in consumables cost by 68%. Sensitivity analysis is used to evaluate the process in different manufacturing scales (50-1200 kg/yr), upstream titers (1.5-5.5 g/L) and downstream yield (70%-80%). Cost savings can be observed through the whole range.

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Monoclonal antibody (mAbs) are used for the treatment of cancers, autoimmune diseases, inflammatory, infectious, and microbial diseases1. mAbs are widely used with four out of the top five therapeutic proteins being mAbs and among all the therapeutic proteins, mAbs count for almost half of the selling amounts 2. It is also predicted that the mAbs market size will be worth $138.6 Billion by the year 2024 1. As a highly demanded biological product, the market and development of mAbs are rapidly grown in recent years – four new products are approved every year and 78 have been approved from 2017 3.

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Process intensification needs to be considered to accommodate the increasing mAbs’ market. Process intensification focuses on developing novel apparatuses and methods, and aims to find more efficient, cheaper, sustainable technologies by reducing production capacity, energy consumptions and waste formations in the process4-5. Using novel technologies such as perfusion production bioreactor and continuous purifications, continuous biopharmaceutical process reduces the equipment footprints multifold compared to the corresponding batch or semi-batch process, and continuous operating mode offers rapid capacity adjustment and process scaling up6. In addition, continuous process improves product quality, reduces bioburden risks and increases volumetric productivity of the process. It also standardizes the biological production by building multiproduct facility which can produce both stable and nonstable proteins6. All the above advantages of continuous biopharmaceutical processing results in process intensification. Biopharmaceutical manufacturing process is in the transition from batch to continuous production7 and there exists FDA approved product produced from continuous upstream bioprocessing8.

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In recent years, a lot of work has been involved in the development of continuous processing for biologics production. In upstream, comparing to batch/fed-batch production bioreactor, perfusion bioreactor maintains higher cell density, cell variability and it increases protein quality and productivity 9. The process is operated with various cell retention devices, such as spin filter, tangential flow filtration (TFF), alternative tangential filtration (ATF). Bielser et al.10 reviewed the general information of perfusion process by discussing the application of perfusion bioreactors and its operating conditions in different types of manufacturing processes and critically analyzing its capability in future development. Voisard et al. 11 and Castilho, Leda R. 12 introduced different cell retention devices and their application in mammalian cell cultures. Pollock et al. 13, Daniel et al. 14 and Chotteau et al. 15 also compared spin filter, TFF, and ATF in terms of titer, productivity, economic benefits, and environmental impacts.

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Introduction

ACS Paragon Plus Environment

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Regarding downstream processing, most of the work focuses on improvement of continuous protein A chromatography which is the most expensive process in downstream batch processing. Multiple methods have been developed and are available by different manufacturers, such as periodic countercurrent chromatography from GE Healthcare Life Science, countercurrent tangential chromatography from Chromatan, Inc., multi-column chromatography from Repligen Corporation, and some other nonchromatography methods are explained in the review by, Jungbauer, Alois 16 and Zydney, Andrew 17. The review also summarized other operations in downstream processing, including tubular reactor, UV reactor for virus inactivation; high-performance tangential flow filtration, flow-through chromatography, simulated moving bed chromatography for polishing; single-pass tangential flow filtration, and cascade diafiltration in continuous formulation. Patil and Walther18 review both upstream and downstream technologies in continuous manufacturing.

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Warikoo et al.19, first successfully demonstrated the integrated process with perfusion cell culture connected to periodic countercurrent chromatography system for monoclonal antibody and recombinant human enzyme production at the development scale. Godawat et al.20 successfully tested the stability of mAbs production in an end-to-end fully continuous process with additional polishing step. A pilot-scale proof of principle process with critical manufacturing unit operations including formulations is integrated successfully from experiment 9, 21-22. Karst et al. 23 reviewed and evaluated continuous processing by product quality. The comparison between batch and continuous processing can be evaluated by using economic analysis. Hammerschmidt et al. 24 compared fed-batch (with fed-batch upstream and batch downstream ), hybrid and fully continuous process by economic analysis. Instead of using continuous chromatography, the continuous process used precipitation for protein purification. Klutz et al. 25 presents a complete cost structure and process decision-making of antibody production in fed-batch, hybrid and continuous processes with plant capacity 200 kgAPI/yr. It also confirms that the hybrid process has benefit comparing to the other two methods from 50 to 1000 kgAPI/yr. Walther et al. 26 applied economic modeling and Monte Carlo modeling on integrated continuous biomanufacturing profits analysis. Pollock et al. 27 evaluated fedbatch, continuous, hybrid processes with the respect of economic, operational and environmental feasibility. Different manufacturing scales (up to 200 kg/yr) with different upstream titers are evaluated in the process.

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In this paper, biopharmaceutical manufacturing efforts towards process intensification are presented and evaluated. A detailed integrated fed-batch (with fed-batch upstream and batch downstream) and continuous processes is developed to produce mAbs from mammalian cells using Superpro designer (Intelligen, Scotch Plains, NJ). The analysis considers material balances, process scheduling, and equipment occupancy charts to enable a comprehensive evaluation of the two alternative production routes. Deterministic cost including different cost categories is utilized to evaluate the improvement of continuous processing over fed-batch in base case scenario. Different scenarios are built using sensitivity analysis to examine the effect of important operating parameters and manufacturing scales to overall continuous process performance.

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2. 2.1

Method Process description

Biopharmaceutical manufacturing for mAbs production contains the following steps: inoculation, cell cultural and protein production, clarification, primary capture, polishing, and final formulation. Figure 1 and Figure 2 provide process operation flowsheets for fed-batch and continuous processes respectively. Inoculation process is applied to cell culture scale up by using a series of cell culture passages 28 such as test tube, T flasks, shake flasks or seed cell culture bioreactor which are shown in Figure 1. The purpose of this step is to grow the cells to certain density and viability before transferring them to production bioreactor. From literature, inoculation cell density ranges between 0.2-1x106 cells/ml 10, 19, 29-31. Some publications show that the density can reach as high as 2-3 x106 cells/ml 15. In this paper, 1.5x106 cells/ml is considered as the peak cell density in inoculation bioreactor. Fed-batch and continuous processes share the similar inoculation bioreactor. Extracellular protein such as mAbs is traditionally produced in fed-batch production bioreactor under temperature 36.5-37 ℃, with dissolved oxygen 30-50%. Production media, antifoam are


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added at the beginning of the operation with adjusted dissolved oxygen concentration, and pH. Then cells from inoculation are transferred to production bioreactor. When the cell concentration reaches 0.7 x106 cells/ml, nutrients, such as glucose, Hy-Soy are added. Nutrients, base, process air and antifoams are added based on the culture requirements. In this way, the bioreactor is actually operated as fed-batch bioreactor with additional nutrient addition. In the fed-batch model, the cell culture can last for 14 days with the titer of 1.5 g/L. The cell density is assumed 5.5x106 cells/ml at the end of the cell culture and productivity is calculated as 0.107 g/L/day with cell specific productivity 37 pg/cell/day. After the fermentation, material from bioreactor is transferred to centrifuge, where biomass and most impurities are removed. Before transferring the product to the primary capture step for further purification, several filtration steps need to be considered to remove the remaining cells in the solution and protect protein A column and resin from contamination and clogging. In industrial manufacturing, purification through protein A column is one of the most important steps in downstream processing, which removes most of the impurities such as host cell protein (HCP), DNA, media component and virus particles 32. The primary capture operation contains the following procedures:

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1) Pre-sanitization and pre-equilibrium: using acidic buffer, alkaline buffer, salt buffer and water to wash and activate resin; 2) Equilibrium: salt buffer sodium chloride and sodium phosphate are applied to adjust pH and prepare for protein loading. This step prevents the protein aggregation and deactivation during loading process; 3) Loading: protein binds to the resin with little amount of impurities during this step, and most of the impurities, water, and unused media is removed in flow through; 4) Washing: salt buffer sodium chloride and sodium phosphate are used to remove unbonded impurities and residuals; 5) Elution: protein product with minor impurities are recovered from resin and transported to the next step by using acid salt; 6) Regeneration and final wash: acid and alkaline buffer are used to remove protein and impurities left inside of column.

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During the primary capture, the column usually runs for several cycles to finish the purification of one batch. This is done since a large amount of protein is produced from the bioreactor, and the capacity of protein A column is limited by resin binding capacity (30 g/L). Due to the protein loss during loading, washing and elution steps, the protein yield of this unit operation is assumed 95%. By adding acid, virus inactivation of elution proceeds in blending tank for 1 hour to prevent virus growth. Alkaline buffer is then added to neutralize the solution pH and followed by diafiltration to prepare for polishing step. Anion exchange chromatography (AEX) working under flow-through mode is used for polishing step to remove the residual impurities, such as DNA, and final formulation is realized by ultrafiltration and diafiltration. In the model, ultrafiltration and diafiltration are operated in the same equipment, and protein concentration is adjusted during these operations. In fed-batch process, pooling tank needs to be used between each unit operations to maintain the process sequencing and free up expensive or time-consuming unit operations 33. Steaming in place (SIP) and cleaning in place (CIP) are also applied and scheduled before and after equipment usage, including seed bioreactor, production bioreactor, storage tanks, virus inactivation tanks, diafiltration, and ultrafiltration. It is need to point out that Figure 1 and Figure 2 only show the main operations in the two processes operations. Pooling tanks, filters are not included in these figures but are considered in the simulation.


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Figure 1: Fed-batch process flowsheet for monoclonal antibody production


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In perfusion bioreactor, high cell densities can be achieved using continuous fresh nutrient fed and continuous product, by-product, and waste removed from bioreactor. Under this mode of operation, the percentage of cell viability, product quality, and volumetric productivity can be improved compared to fedbatch process. It is reported that cell density of perfusion bioreactor can reach 20-60x106 cells/ml and most cell density of perfusion reactions are within this range9, 13, 15, 19, 25, 27, 31, 34. Walther et al. 26 achieved density as high as 100x106 cells/ml with an annual production rate of 1236 kg/yr. The perfusion bioreactor viable cell density chosen for this paper is 60x106 cells/ml with volumetric productivity of 1.68 g/L/day and cell specific productivity 28 pg/cell/day. Chotteau et al. 15 found that the product quality varies during the early stage of the bioreactor culture and productive stage. The product harvests should be started a few days after the system reaching cell density target. However, the time should be balanced against the overall productivity in manufacturing process. In this, 4 days are considered as the setup time before the cell cultural reaching steady state. However, the protein lost during this period is not considered. 0.02 um Tangential Flow Filtration (TFF) is connected to perfusion bioreactor which is used to separate cells from liquid product and return cells to fermentation. Cell bleeding which is used to maintain cell density in perfusion bioreactor is not included in this system and the cell density is assumed constant. The fermentation lasts for 24 days even though some papers use up to 60 days 13, 19, 26. Filter clogging, and fouling may occur during perfusion so maintaining proper operation can reduce the failure of bioreactors. Similar to batch processes, filters are also added before the periodic countercurrent protein A column (PCC) for primary capture. Blending storage tank is designed to distribute the product to different columns. Four columns work in parallel which mostly increase the resin usage and reduce the protein loss and buffer consumption. The resin binding capacity for PCC process is 60 g/L. A pooling tank is installed right after the protein A column to collect the protein before transferring the product to virus inactivation. Two virus inactivation tanks work alternatively i.e. one tank is working under acidic inactivation while the other is cleaning. Two static mixers and pH adjusters are applied before and after the inactivation tanks. Similarly, two AEX columns also work alternatively to achieve continuous polishing. Before the polishing step, countercurrent staged defiltration is applied for continuous buffer exchange with 5 diafiltration volumes which replace nearly all of the buffer and salt by sodium phosphate 17. Nanofiltration removes virus in the product. Protein is further concentrated by using staged single pass tangential flow filtration (SPTFF) and buffers are exchanged by using countercurrent staged diafiltration. The lifetime of membrane in filtration is assumed to be 10 cycles. Casey et al. 35 applied high concentration SPTFF cycles testing and approved that the ultrafiltration can complete 10 cycles successfully and concentrate IgG from 45 g/L to 200 g/L. In this paper, the continuous system only requires protein concentrate from 11 g/L to 30 g/L. Thus applying 10 cycles lifetime might result in an over estimation of the consumable cost. Since all the processes operate in a continuous mode under lower flow rate, no large pooling tanks are needed between unit operations. The most important operating parameters are summarized in Table 1. The maximum operating time of the two processes is 330 days/yr.


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Figure 2: Continuous process flowsheet for monoclonal antibody production


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Table 1 Operating conditions Fed-batch Processing 1.5x106 cells/ml 5.5x106 cells/ml 15000 L

Inoculation cell density (seed) Bioreactor cell density (production) Max bioreactor volume set Perfusion rate Downstream yield Cell cultural time The annual number of batch Annual production rate

75% 14 days 48 618 kg/yr

Continuous Processing 1.5x106 cells/ml 6 x107 cells/ml 3000 L 1 vvd 75% 24 days 9 624 kg/yr

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In downstream process, the yield of each unit operation is shown in Table 2. The yield for both batch and continuous downstream process is 75%.

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Table 2 Downstream unit operations yield Clarification Batch Continuous

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99% 99%

Primary capture 96% 98%

Virus inactivation 95% 95%

Diafiltration

Polishing

96% 91%

95% 95%

Virus removal 98% 98%

Final filtration 95% 96%

Equipment occupancy charts are shown in Figure 3 and Figure 4. One equipment is chosen to represent each unit operation to simplify the results’ presentation. For example, in Figure 3, the inoculation bioreactor is shown to represent inoculation process while the inoculation flasks, and tubes are not shown in the chart. In fed-batch process, the operation starts right after the previous operation finished. In continuous process, all the major steps of operations start almost at the same time, except the inoculation and CIP and SIP processes. The annual production rate can be reached by using multiple equipments in fed-batch process. It is found that, process bottleneck of fed-batch process is in upstream, thus, two seeds bioreactors and four production bioreactors work in parallel and each of those bioreactors start a new batch to reduce the time between consecutive batches. In this case, the number of equipment utilization between fed-batch and continuous processes is different. Scheduling is also applied to adjust the order of each procedure in different unit operations. For example, in both fed-batch and continuous processes, product transferring in task is manually linked to the product transferring out task from previous operation. In continuous process, the operation needs to be performed continuously under the same time period as the previous operation. CIP and SIP are manually adjusted so that maximum numbers of operations can be fitted to the minimum amount of CIP and SIP units.

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Figure 3: Equipment occupancy chart for the fed-batch process (SBR: inoculation bioreactor, BR: production bioreactor, DS: centrifuge, C-101: protein A column in primary capture, V104 and V111: virus inactivation tank, DF-101: diafiltration, C-102: AEX column, DE: virus removal, DF-102: diafiltration and ultrafiltration)

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Figure 4: Equipment occupancy chart for the continuous process (SBR: inoculation bioreactor, BR: production bioreactor, DS: centrifuge, C-101: one protein A column in primary capture, V110: virus inactivation tank, UF-111: diafiltration, C-104: AEX column, DE: virus removal, UF-101: ultrafiltration, UF-107: diafiltration)

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2.2

Economic analysis method

Total capital investment and operating cost are two major costs in economic analysis. Total capital investment which includes direct working capital, startup and validation costs, and direct fixed capital cost, is calculated based on equipment expenditure. Direct fixed capital contains equipment purchase cost, installation, piping, instrumentation, insulation, electrical, buildings, auxiliary facilities, engineering, construction cost as well as contractor’s fee and contingency. Each cost is estimated by multiplying a certain coefficient to equipment purchase cost as listed in Table S1 in supporting information following the work of Harrison et al. 36The cost of equipment is evaluated based on literature and vendor resources. Working capital represents the assets that must be available to ensure the facility processing and includes 1 month of labor, raw material cost, utilities and waste treatment cost. Scaling rate is applied to estimate capital cost as shown in Equation (1). The most important prices and parameters that are used in economic analysis are listed in the Table S2.


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C2 = C1 ∙ (

size2 a ) size1

(1)

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The operating cost contains material, consumables, utilities, labor-depended, quality control (QC) and quality assurance (QA) cost and facility dependent cost (including the cost related to use of facility: equipment maintenance, depreciation of the facility and miscellaneous cost). Material and consumables costs are referenced from literature and online vendor resources. QC/QA cost is estimated as 15% of total labor cost. The total operating cost of the product ($/g) is also named as cost of goods per gram (COG/g) and it is usually used to calculate operating cost per unit of production. In this paper, we redefine the COG/g as shown in Equation (2) and consider both total capital investment and operating cost.

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Total capital investment $ $ (2) ( ) + Annual operating cost( ) Years of operation yr yr COG = g Annual production rate ( ) yr Regarding the selling price, the prices of monoclonal antibodies that are approved by the FDA from the year 1997 to 2016 are shown in Figure 5 37. It can be noticed that most of the prices are below 100 $/mg. 20 $/mg is assumed as selling price in base case scenario which represents the average selling prices of drugs below 100$/mg.

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Figure 5: Monoclonal antibody price approved from 1997 to 2016 (The figure is plotted based on literature data) 37

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Sensitivity analysis is also used to evaluate the effect of different parameters considered in simulating the continuous process. Parameters considered are the upstream titers, downstream yields and manufacturing scales to the overall process economic benefits. In fed-batch and continuous processes, most facilities are interconnected and the operating time of one procedure is scheduled depending on another process. Due to the complexity of the process, different scenarios are designed for process evaluation. For upstream titer analysis, facility size is kept constant while titer varies from 1.5 g/L to 5.5 g/L. The downstream yield in the base case scenario is 75%. Different scenarios with yield of 80%, 85%, and 90% are analyzed for fed-batch and continuous process. For plant capacity analysis, the facility size varies from 50 g/L to 1200 g/L and all the other operating conditions are kept constant.

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2.3

Computational platform used

Three simulation software packages have been commonly used for cost analysis: SuperPro Designer (Intelligen, Scotch Plains, NJ), Bio-Solve (Biopharm Services, UK), and Aspen Batch Process Developer (Aspen Technology, Burlington, MA). Bio-Solve is the most widely used in economic analysis on continuous biopharmaceutical processing. It contains yearly updated built-in cost data library and available for COG/g


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analysis, cash flow analysis, multi-process comparison 38 in all types of biotech products 39. The software is the Excel-based tool and benefits from all Excel add-in’s such as using Crystal Ball by Oracle Corp for tivariate analysis and variability simulations 39. However, the Excel-based simulation tool is time consuming in multi-product analysis and resource constrained facilities 40.

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Aspen Batch Process Developer is a recipe-driven simulator that can be applied both in process evaluation and process economic analysis. Material balance, facility fit and design, scale-up and scale down, detailed economic analysis can be obtained from the software. Combining with Aspen Plus Dynamic, both the steady state and dynamic of the process can be analyzed. However, the software is limited to process design in batch and continuous chromatography.

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SuperPro Designer is a recipe-driven simulation tool that can be used to both batch and continuous integrated biopharmaceutical manufacturing process modeling. By adding a sequence of tasks in each unit operations, material and energy balances can be calculated and tracked through the whole process. Users have an option to choose either the rating or design mode in their process simulation. Under rating mode, the design parameters of equipment can be set, and different products or different production rates can be tested in the model for facility fit. Under the design mode, the maximum equipment dimensions can be set and the simulator can automatically calculate the design parameters for users. Process debottlenecking for example reducing the starting time between two unit operations can also be achieved in the simulation tool. The software provides equipment occupancy chart, gantt chart, material consumable chart labor required chart and throughput analysis to track and analyze detail of process operation. SuperPro designer has the capacity for business modeling, such as economic analysis, cost of goods analysis and essential parameters can be adjusted within the model or transferred to Excel for further analysis.

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Since Aspen Batch Process developer is specific on batch process and continuous chromatography and Bio-Solve is specific on economic analysis, SuperPro designer provides flexibility on process design, facility analysis and process scheduling on both batch, continuous and semi-continuous processing which is the best fit for both process and economic analysis. Thus, the model in this study is built in SuperPro designer simulator, which contains detailed material balance, cleaning methods and equipment scheduling. Economic analysis and throughput analysis are also performed in the Superpro designer for process evaluation.

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3 3.1

Results and Discussions Cost comparison: fed-batch and continuous processes

This section provides the comparison between fed-batch and continuous processes based on the base case scenario where the production rate is adjusted to 620 kg/yr and selling price of mAb is assumed $20/mg based on the analysis in Figure 5. The analysis includes capital investment cost and operating cost with cost breakdowns for both operating modes. The result shows that the overall capital cost in fed-batch process is $165 million, which is 3 times that of continuous process ($53 million). The real plant cost can exceed the cost calculated due to the levels of details considered in the process, for example, buffer preparation tanks and transfer panels that are not built in the model. The operating cost of fed-batch and continuous processes are $61 million/yr and $32 million/yr, respectively. The overall cost of fed-batch mode is $99/g and that of the continuous mode is $61/g. Under the assumption of 15 years’ project with 3 years’ facilities construction and with 7% interest, the net present value difference between fed-batch and continuous process is $720 million.


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Figure 6: Cost of each unit operation in fed-batch process

Figure 7: Cost of each unit operation in continuous process

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Figure 6 and Figure 7 show the cost of different unit operations in fed-batch and continuous processing, respectively. The upstream cell culture unit operation contributes as high as 51% of the overall batch process and inoculation contributes 22%, which are mainly due to the high capital cost of the two unit operations. In order to reduce the time between consecutive batches and achieve 620 kg/yr annual production rate, 2x2 inoculation bioreactors (2 different bioreactors and 2 pieces each) are used in inoculation operation and 4 production bioreactors are used in cell culture. Labors are also scheduled to each of the bioreactor. With multiple inoculation bioreactors and 15000L bioreactors, the facility dependent and labor cost result in 74% cost for the inoculation operation and 63% for the cell culture. Comparing to fed-batch process, continuous process reduces the number of bioreactors to two inoculation bioreactors and 1 perfusion production bioreactor. In addition, the size of production bioreactor also decreases from 15000L to 2800L. Thus, the facility dependent cost and labor cost in continuous process significantly decreases in comparison to fed-batch. As a result, the upstream cost in continuous process is reduced to one half of original cost. However, upstream unit operations still occupy the highest portion of overall cost. Material costs are another major cost in upstream unit operation in both processes which take 31% of the cell culture cost in fed-batch process and 75% of that in continuous. This is because of low volumetric productivity (0.107 g/L/day) and low culture medium conversion rate in fed-batch bioreactor. In continuous process, the volumetric productivity increases to 1.68 g/L/day. However, due to the usage of same culture medium, it assumes that there is no significant increasing of medium conversion rate in perfusion process. From the simulation result, the material cost in perfusion process does not account to significant savings to the overall cost.

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Continuous processing not only has benefit in upstream operations but also in downstream operations. Figure 8 compares the COG/g of different unit operations in fed-batch and continuous processing. Continuous process reduces the primary capture by two folds, which accounts for the highest portion of the downstream cost in both operating modes. In the primary capture step, the size of protein A column reduces from 286L to 4 columns of 3.14L which decreases the capital cost and facility dependent cost. The PCC system has internal connection between the protein A columns, which improves the protein yield and resin utilization during this step. Thus, material cost from equilibrium, washing, elution and regeneration buffers is also reduced. In addition, the PCC step uses more expensive resin but provides higher resin binding capacity and life cycles, which compensates the high unit price. Above all, most savings are achieved from the cost reduction of protein A resin which is only 28.4% of that in fed-batch process.


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Cost of goods ($/g)

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Pimary capture

Virus Diafiltration Polishing inactivation Batch

Virus removal

Final filtration

Continuous

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Figure 8: COG of fed-batch and continuous upstream downstream unit operation

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However, not all the unit operations in continuous process contribute to cost savings over fed-batch process. Virus inactivation and polishing operations have higher COG/g than batch process as shown in Figure 8. In virus inactivation process, two tanks work alternatively in a way that as one tank is under cleaning, the other one is working under low pH virus inactivation. Similarly, two AEX columns are used in polishing step. In these two steps, there is no significant improvement of process operations. Although the size of equipment decreases in both unit operations, labor cost increases due to requirement of continuous operations. There are also no significant savings of final filtration step in continuous process. The final filtration contains not only the ultrafiltration and diafitration steps but also sterilize filter and final packaging, so that additional labors are assigned in this process. Thus, labor cost of filtration in continuous process is higher than that of batch. However, material cost in this operation is reduced which compensates the labor cost, thus, no significant savings can be observed. Figure 9 shows the cost comparison between fed-batch and continuous processes for all different cost categories. The main benefits of continuous process are due to capital cost investment and facility dependent cost, which are caused by the smaller footprint of all the unit operations through the process. In addition, large storage tanks are not used in continuous processing 22 that results on lower operating and capital cost. Consumables cost is also reduced by the resin cost savings in primary capture operation as explained in the previous paragraph. Even though continuous process shows savings on material and labor cost, the savings are not as apparent as that of facility dependent cost and consumables cost. In fed-batch process, each equipment is assigned one or two units of labors and is assumed that labor is needed only at the time of operation. In the continuous process, considering the automations used in the system, one or two labors are assigned to each unit operation that contains many equipment. However, the continuous process is running continuously, which means multiple labor units have to be occupied from start to the end of the process with shifts. Material cost is mainly due to upstream cell culture process. Perfusion method provides a capability of upstream operation with wilder utilization of cell lines and continuously adding culture medium. Thus, improving the conversion or utilization of medium by using different cell lines or medium will reduce the material cost per gram of product31, 41-42.


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Figure 9: Cost of goods analysis between fed-batch and continuous

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The throughput analysis is used to evaluate the plant capacity effect on production cost in fed-batch and continuous processes as shown in Figure 10, and the capacity ranges from 50 kg/yr to 1200 kg/yr. It shows that as the plant capacity increases, the unit operating cost ($/g) of product decreases both in fed-batch and continuous processes. In both operating modes, the labor dependent and QC/QA cost decrease significantly as plant capacity increases. This is mainly due to labor dependent cost, which is scale independent. With annual production rate increasing, the averaged labor dependent cost per unit of protein production decreases. For example, the labor dependent cost in continuous process ($6.51 million) remains constant in all plant capacities. However, as the throughput increases from 50 to 1200 kg/yr, the unit labor dependent cost decreases from $130.14/g to $5.50/g. Furthermore, facility dependent cost which depends on labor cost and capital cost also decreases as shown in Figure S1 and Figure S2. It also shows that, consumables, QC/QA costs decreases. There is material cost savings which is not obvious, and the main reason is that the material cost is dominated by cell culture medium which is independent to the plant capacity.

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Comparing to the fed-batch process, the benefit of the continuous process becomes less significant as plant capacity increases. As explained in the previous paragraph, the cost savings are mainly from facility dependent cost and labor cost. The operating costs of different unit operations that are dominated by these two types of costs will be reduced the most. From Figure 11, upstream cost savings are more significant in fed-batch process and downstream cost savings are more significant in continuous process. However, among the total cost, upstream cost takes up a higher portion than downstream. Thus, the cost saving from fed-batch process is more significant as the plant capacity increases.

Sensitivity analysis

The parameter fluctuations and variations are very common in biopharmaceutical manufacturing. For example, upstream titers are varied by different cell lines and media used. Consumptions, such as resin and membranes, their life cycles, membrane capabilities, yields and costs are changed with market perturbation and types of product produced. Thus, uncertainty and sensitivity analysis can be used to investigate the impact of variable parameters inputs on the final result 43. In this section the analysis is focused on the uncertainty and sensitivity analysis of plant capacity, upstream titer and downstream yield on the overall economic analysis.

Throughput analysis


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Figure 11: A comparison between fed-batch and continuous cost on unit operations among the different plant capacity

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Titers represent the concentration of protein product harvested from production bioreactor. Development of upstream process, improvement of seed bioreactor, and the choices of cultural media and cell lines increase the titers in bioreactors which will improve the upstream productivity, and this will affect downstream operating cycles, times and consumables used. Due to the number of interconnections and scheduling among the equipment in fed-batch and continuous system, increasing the upstream titer will cause process rescheduling and system conflicts. Thus, multiple scenarios with different titers are designed for the upstream titer analysis.

Upstream titer analysis


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In Figure 12, the simulation results for fed-batch and continuous processing are compared under different upstream titers in different scenarios: 1.5 g/L (scenario 1), 2.5 g/L (scenario 2) and 5.5 g/L (scenario 3). It shows that, with increasing titer, the overall operating cost per unit product ($/g) decreases in both operating modes. With titer ranging from 1.5 g/L to 5.5 g/L, the operating cost ($/g) of fed-batch process is reduced by 58%, and the continuous process is reduced by 50%. This is due to the increasing annual production rates of the two operation modes, shown in Table 3. Under the same upstream operating conditions, the increase of upstream productivity shifts the bottleneck of the process from upstream to downstream. Since facility sizing remains constant through all scenarios, to purify more protein, more cycles need to be used in major purification operations. From scenario 1 to scenario 3, the fed-batch upstream productivity increases from 17.1 kg/batch to 62.7 kg/batch, and the number of protein A column cycle increase from 2 cycles to 7 cycles and the unit operating time increases from 0.62 days to 1.83 days. To prevent product overflow, hold vessels that are used between unit operations, and virus inactivation tanks need to be operated in cycles. Thus, the overall annual batch numbers decrease from 48 cycles to 45 cycles. Similarly, in continuous process, with titer increasing, primary capture step is also the bottleneck. Since upstream production rate increases from 109 kg/batch to 398 kg/batch, the PCC cycle numbers increase from 127 cycles to 468 cycles, which causes the operating time to increase from 24 days to 73 days. Thus, the overall working cycles reduce from 9 batches to 3 batches. For this case, continuous processing has less annual protein production rate, so cost savings are not as significant as for the fed-batch process.

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Table 3 Annual production rate of fed-batch and continuous process with different upstream titers Upstream titer 1.5 g/L 2.5 g/L 5.5 g/L

Fed-batch production rate (kg/yr) 618.16 987.00 1227.00

Continuous production rate (kg/L) 620.00 776.98 856.58

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Figure 12: A comparison between fed-batch and continuous operating cost with different upstream titer

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Even though cost savings in fed-batch process becomes more significant as titer increases, the operating cost per unit of fed-batch product is still higher than that of continuous. This is mainly due to facilitydependent cost and consumables cost of continuous operations being 3 times lesser than that of fed-batch. It is also interesting to show that as titer increases, the cost ratio between upstream and downstream


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increases in fed-batch operations but decreases in continuous operations. It indicates that, continuous downstream unit operations are more sensitive to the titer in upstream and as titer increases, the process bottleneck will shift from upstream to downstream. To improve the productivity and flexibility of continuous processing, multiple downstream facilities should run in parallel or larger facilities need to be used. Since continuous primary capture uses 4 columns of 3.14 L, either adding another set of PCC operation or increasing the column sizes is cost effective. Thus, the operating cost of continuous process can be further reduced. Hammerschmidt et al. 24 considered fed-batch plus the chromatography system and perfusion plus the precipitation system and compared the cost of goods for different upstream titer. The results showed that for the fed-batch system, as titer increased, the ratio between upstream and downstream decreased which means the cost shifts from upstream to downstream. However, this result is opposite to what we obtained. It needs to be highlighted that, the facility size in Hammerschmidt et al. analysis is varied with titer while it is kept constant in this paper. By varying facility sizes, increasing the upstream titer also shifts operating cost from upstream to downstream in fed-batch and continuous processes.

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Different operating scenarios are designed to analyze downstream yield effect on operating cost in both fed-batch and continuous processes. Downstream yield in base case scenario is 75% and downstream yield variations range from 70% to 80% while the facility sizes are kept constant. In fed-batch and continuous processes, different downstream unit operations have different yield. In order to decrease the overall yield, among all the downstream unit operations, highest operation yields are decreased. Similarly, in order to increase the yield, lowest operation yields are adjusted and increased.

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As downstream yield increases, the overall operating cost decreases, shown in Figure S3. When yield increases, the annual production rate increases. Thus, the utilization of equipment increases and the unit operating cost increases. However, when the operating yield reaches 80%, the size of holding tank in fedbatch process is not large enough to maintain the operating volume. Here, the number of operating cycles is increased and the total operating time per batch also increases. Thus, the cost savings in operating cost from downstream yield 75% to 80% is not as much as that from 70% to 75%. In continuous process, cost savings can be observed with an increase in downstream yield. However, there are no obvious changes or cost shifts between upstream and downstream. The cost savings of continuous process decreases is not compared to fed-batch process, which shows that the continuous process is not as sensitive as fed-batch.

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Downstream yield

Conclusion and future work

This paper highlights efforts in biopharmaceutical manufacturing towards process intensification highlighting the advantages of alternative process units and continuous operating mode. It provides a comprehensive comparison of traditional fed-batch process as it compares with intensified continuous operation in biopharmaceutical manufacturing by using economic metrics and sensitivity analysis. From base case scenario, with 620 kg/yr annual production rate, continuous process, with small footprint and high automations, reduces capital cost, facility dependent cost and labor cost significantly compared to fedbatch process. Consumables and material costs of continuous process are also reduced, mainly because the resin utilization in continuous process is increased, and with small footprint, the buffer usage for downstream cleaning and primary capture is also reduced. From sensitivity analysis, scenarios are designed to compare fed-batch and continuous processes with different operating conditions, including, various plant capacities, upstream titers and downstream yield. Results show that, as plant capacity increases (from 50 kg/yr to 1200 kg/yr), operating cost in both fed-batch and continuous processes decreases and cost in continuous process is lower than that of fed-batch process in all ranges. Similarly, as titer increases or downstream yield increases, the operating costs also decreases. Increasing upstream titers shifts the process bottleneck from upstream to downstream, especially primary capture step, which causes longer downstream operating time. In continuous process, adding another downstream line or increasing size of primary capture column can resolve bottleneck cost-effectively.


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Several assumptions are made in this model to simplify the process and calculations. In upstream process, same inoculation operation is used in both fed-batch and continuous processing. In recent years, researchers have been working on inoculation operation by using N-1 perfusion inoculation bioreactor that has high-density seeding, followed by fed-batch bioreactor. The method may increase the inoculation cell density, reduce the production bioreactor fermentation time and improve the production capacity and product quality 10, 44. In addition, most of the cleaning recipes in continuous process are modified and similar actions are done in the fed-batch process. Total operating times per batch in continuous process and cycle numbers are calculated based on unit operation running time but not optimized. Failure rate and contaminations are not considered in the production bioreactor section in this work. Since perfusion bioreactor contains external filters and cells are recycled back to the fermenter, the contamination chance in perfusion process should be higher than that of fed-batch process. Filter clogging, and fouling can also cause process failure. However, when process failure happens, the whole batch of product needs to be disposed in the fed-batch processing, however in continuous process, only the product affected by the failure should be discarded.

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Instead of economic benefits, continuous biopharmaceutical processing shows advantages in the area of production system standardization, process flexibility, scaling, product quality, and it is available to use single-use technology. The development of continuous process is also under a good regulatory condition. In 2011, US Food and Drug Administration’s strategic plans45 pointed out that the continuous manufacture of biologicals is encouraged46 and the product quality can be improved by using Quality by Design (QbD). FDA has also confirmed that the continuous process offers advantages in development and manufacturing47. Continuous biopharmaceutical processing allows rapid capacity adjustment by adjusting the number of parallel production lines6. The duration of production can also be changed based on the requirement of certain product. Since the continuous processing reduces the total time that product stays inside of production line, especially in the bioreactor, both stable and non-stale protein product can also be produced in multiproduct continuous facilities so that implement process standardization. More important, the decreased residence time in bioreactor and elimination of intermediate hold steps improve the product quality by reducing the risk of product degradation and post secretion enzymatic modification. With the uniformed microenvironment control, the continuous processing also minimizes the product variability48. In addition, scales in continuous processing are equivalence for pilot, clinical and commercial production, which increases the speeding of scale up and minimizes the technology transfer. However, challenges are also come along with those benefits. The integrated fully continuous processing requires high degree of automation with minimal operator involvement. While different vendors share different control system which increases integration difficulty6.

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For future work, the continuous process need to be optimized, with respect to the batch time, number of operating cycles, process lines and facility sizes, with consideration of process failure rate. Single-use methods also need to be involved in the analysis. In single-use system, cleaning such as CIP and SIP do not need to be used which reduces buffer and energy consumption. However, the single-use equipment needs to be replaced and disposed. In addition, it also has the limitation of facility sizes, for example, production bioreactor is limited under 2000 L. Thus, including all the above considerations, a more realistic and cost-effective integrated continuous process model can be built.

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Acknowledgment

The authors would like to acknowledge financial support from NSF under grant 1547171.

Supporting information statement

This information is available free of charge via the Internet at https://na01.safelinks.protection.outlook.com/?url=http%3A%2F%2Fpubs.acs.org%2F&data=02%7C 01%7Cmarianth%40soe.rutgers.edu%7C1d4f32efb67848740a3008d6563d4fad%7Cb92d2b234d354470 93ff69aca6632ffe%7C1%7C1%7C636791218679113281&sdata=MgS52eVCy3o0MbcnAGGkW0bd TFbTD1mQmD%2BPJjUQ0r0%3D&reserved=0.


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Economic analysis capital cost estimation; unit price of equipment, consumables, disposable; fed-batch plant capacity analysis with operating cost categories; Continuous plant capacity analysis with operating cost categories; fed-batch and continuous COG ($/g) comparison with different downstream yield

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Comparison between Batch and Continuous Monoclonal Antibody

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