Economics of fed-batch operation: a computer-aided approach

The widely anticipated economic potential of fed-batch operation was quantified for a therapeutic product by flowsheet simulation. A process for production of t-PA from CHO cells based on fed-batch operation was compared to a base process that operates in batch mode. Two cases of fed-batch operation were considered, case A, where the product concentration was assumed to be four times the concentration obtained with base process and case B, where the concentration was eight times. The simulator, Bioprocess Simulator (BPS) from Aspen Technology, reported the final bioreactor volume and the total amount of the continuous feeds added during the fed-batch operation. BPS was also used to simulate the downstream processes. Comparison of the economic performances of the processes revealed that return on investment (ROI) of the base process would increase by 112% by switching to case A fed-batch operation from batch mode. Case B, on the other hand, would result in an increase of 288%. The importance of downstream processing for recovery of high-value products became apparent from this study. A breakdown of equipment purchase cost showed that the contribution from the product recovery section increased from 62% for base case to 77% for case A as the product concentration increased by fed-batch operation. For a fixed recovery of 40%, contribution from the downstream section was found to decrease to 70% for case B compared to case A. It was concluded from the results that higher product concentration would not result in proportionate increase in ROI because of limitations in the recovery section. A sensitivity analysis was carried out on several uncertainties of the simulated fed-batch process.     

Economic comparison of diagnostic antibody production in perfusion stirred tank and in hollow fiber bioreactor processes

The total operating costs of small-scale monoclonal antibody production were calculated for two different upstream options and general downstream procedure based on protein A chromatography. The upstream options were a spin-filter equipped stirred-tank bioreactor (STR) and a hollow fiber bioreactor (HFB). Both the bioreactors were operated in perfusion mode. The total operating costs of the processes were 6,900 €/g for STR option and 6,400 €/g for the HFB option. In the both systems, the costs were dominated by expenses derived from the downstream section (almost 80%) that was almost identical in the both systems. In the upstream section, the investment depreciation was the largest cost item. The lower total costs of the HFB option were a result of lower investment costs and more concentrated product that led into savings also in downstream section. This study brings out the HFB as on viable alternative for stirred-tank bioreactor, especially in small-scale diagnostic monoclonal antibody production.     

Downstream process synthesis for biochemical production of butanol, ethanol, and acetone from grains: generation of optimal and near-optimal flowsheets with conventional operating units

Manufacturing butanol, ethanol, and acetone through grain fermentation has been attracting increasing research interest. In the production of these chemicals from fermentation, the cost of product recovery constitutes the major portion of the total production cost. Developing cost-effective flowsheets for the downstream processing is, therefore, crucial to enhancing the economic viability of this manufacturing method. The present work is concerned with the synthesis of such a process that minimizes the cost of the downstream processing. At the outset, a wide variety of processing equipment and unit operations, i.e., operating units, is selected for possible inclusion in the process. Subsequently, the exactly defined superstructure with minimal complexity, termed maximal structure, is constructed from these operating units with the rigorous and highly efficient graph-theoretic method for process synthesis based on process graphs (P-graphs). Finally, the optimal and near-optimal flowsheets in terms of cost are identified.     

Shortfall of equipment for neonatal intensive care and the introduction of budget holding contracts.

As adequate allowance must be made for the costs of purchasing, maintaining, and updating equipment during the development of contracts the current standing of neonatal units with regard to available equipment was assessed. Data were collected as part of a one year prospective survey of the 17 perinatal units in the Trent region. Adequacy of provision of equipment for recognised intensive care cost was assessed using the recommendations of the British Paediatric Association and British Association of Perinatal Paediatrics. It was assumed that units without recognised intensive care cost had to be able to equip one cot to a standard of intensive care level 1 in the short term. Equipment more than 5 years old was considered likely to warrant replacement or major maintenance within the next two years. With these guidelines over 600,000 pounds would be required to provide sufficient equipment for all recognised level 1 intensive care cost and to allow units without funded cost to provide this level of care in the short term and to replace existing equipment more than 5 years old for these cost alone. This amount could be reduced by 25% by subdividing intensive care cost into levels 1 and 2, thereby reducing equipment requirements, but this would impair the units'' ability to perform level 1 care at funded provision, which has already been shown to need expansion. Neither figure takes account of equipment requirements for infants requiring special care. In addition, no allowance has been made for purchase or update of ultrasound scanners or blood gas analysers. If the government''s proposed reforms are to be implemented clinicians need to revise guidelines regarding essential equipment, and plans must be made to correct any existing shortfalls so that they do not become inherited financial liabilities for future budget holders.     

Single-stage chromatographic clarification of Chinese Hamster Ovary cell harvest reduces cost of protein production

A single-stage clarification was developed using a single-use chromatographic clarification device (CCD) to recover a recombinant protein from Chinese Hamster Ovary (CHO) harvest cell culture fluid (HCCF). Clarification of a CHO HCCF is a complex and costly process, involving multiple stages of centrifugation and/or depth filtration to remove cells and debris and to reduce process-related impurities such as host cell protein (HCP), nucleic acids, and lipids. When using depth filtration, the filter train consists of multiple filters of varying ratios, layers, pore sizes, and adsorptive properties. The depth filters, in combination with a 0.2-micron membrane filter, clarify the HCCF based on size-exclusion, adsorptive, and charge-based mechanisms, and provide robust bioburden control. Each stage of the clarification process requires time, labor, and utilities, with product loss at each step. Here, use of the 3M™ Harvest RC Chromatographic Clarifier, a single-stage CCD, is identified as an alternative strategy to a three-stage filtration train. The CCD results in less overall filter area, less volume for flushing, and higher yield. Using bioprocess cost modeling, the single-stage clarification process was compared to a three-stage filtration process. By compressing the CHO HCCF clarification to a single chromatographic stage, the overall cost of the clarification process was reduced by 17%–30%, depending on bioreactor scale. The main drivers for the cost reduction were reduced total filtration area, labor, time, and utilities. The benefits of the single-stage harvest process extended throughout the downstream process, resulting in a 25% relative increase in cumulative yield with comparable impurity clearance.     

Enzymatic production of cyclodextrins from milled corn starch in an ultrafiltration membrane bioreactor

Cyclodextrin glycosyltransferase (CGTase) was found to be severely inhibited by cyclodextrins. In order to increase the conversion yield by reducing product inhibition and reuse the CGTase in the production of cyclodextrins from milled corn starch, an ultrafiltration membrane bioreactor system was employed. In a batch operation with ultrafiltration, the conversion yield was increased 57% compared with that without ultrafiltration. Operating conditions for the continuous production of cyclodextrins in the membrane bioreactor were optimized by taking into consideration the filtration rate and the conversion yield as follows: initial starch concentration, 7% (w/v); starch feeding rate, 240 mg/h; CGTase loading, 350 units/initial gram starch. When cyclodextrins were continuously produced in the membrane bioreactor under optimized conditions, 340 units of CGTase was require to produce 1 g of cyclodextrins for 48 h, while in the case of conventional batch operation, 1 g of cyclodextrins was produced for 24 h by 1410 units of CGTase. (c) 1993 John Wiley & Sons, Inc.     

膜生物反应器的研究进展

膜生物反应器是近年来发展的废水处理新技术,具有活性污泥浓度高、污泥龄长、占地面积小、投资省的特点。利用膜生物反应器进行污水处理不仅可以大大节约水资源,还可以大大节约能源,节省设备和运行费用,已成为二十一世纪研究热点。膜生物反应器是通过高效膜分离技术与活性污泥相结合,增大污泥中的特效菌来加快生化反应速率,提高废水处理效果。目前处理对象已从生活污水扩展到高浓度的有机废水和难降解的工业废水。本文综述了膜生物反应器在废水中的应用研究情况,并分析比较了各种膜材质的特点、适用范围以及膜的污染因素和清洗方法,展望了膜生物反应器的应用前景及进一步研究方向。     

Tissue plasminogen activator (t-PA) production by human fibroblasts using a bioreactor with t-PA adsorption column

Production of t-PA by human embryonic lung fibroblasts, IMR-90 cells, is regulated by negative feedback control. The increase in the concentration of the extracellular t-PA lead to a reduction of the production. Therefore, we investigated the application of t-PA adsorption column to ceramic bed reactor to promote t-PA production. Amberlite XAD-8 was selected out as an adsorbent, because it is autoclavable and can adsorb 32,000 IU of t-PA per g wet gel. The t-PA adsorption column was located in the medium recirculation line to the vessel. On the other hand, medium was recirculated between the ceramic bed reactor and the vessel using another flow line. The bioreactor system with the adsorption column was about 2.5 times higher with the resulting cumulative t-PA than that without the adsorption column.     

Production of dextransucrase by Leuconostoc mesenteroides immobilized in calcium-alginate beads: I. Batch and fed-batch fermentations

In batch fermentation Leuconostoc mesenteroides immobilized in calcium alginate beads produced a total dextransucrase activity equal to about 93% of that by free, suspended bacterial cells under comparable conditions in a bubble column reactor. Continuous sucrose feeding (5 g/L h) to the immobilized-cell culture in the airlift bioreactor increased production of enzymatic activity by about 107% compared with ordinary batch operation of this reactor. About 14% of the enzymatic activity produced by the immobilized cells appears as soluble activity in the cell-free broth compared with about 40% in case of free cells. In an airlift bioreactor, both the soluble and the intact (sorbed and entrapped) enzymatic activity produced by the immobilized bacterial cells was about 34% greater under automatic pH control, compared to that produced in a bubble column reactor with only manual pH control. During formation of dextran by intact enzyme within cells and beads, declines are observed in apparent enzymatic activity.     

Integrated two‐liquid phase bioconversion and product‐recovery processes for the oxidation of alkanes: Process design and economic evaluation

Pseudomonas oleovorans and recombinant strains containing the alkane oxidation genes can produce alkane oxidation products in two‐liquid phase bioreactor systems. In these bioprocesses the cells, which grow in the aqueous phase, oxidize apolar, non‐water soluble substrates. The apolar products typically accumulate in the emulsified apolar phase. We have studied both the bioconversion systems and several downstream processing systems to separate and purify alkanols from these two‐liquid phase media. Based on the information generated in these studies, we have now designed bioconversion and downstream processing systems for the production of 1‐alkanols from n‐alkanes on a 10 kiloton/yr scale, taking the conversion of n‐octane to 1‐octanol as a model system. Here, we describe overall designs of fed‐batch and continuous‐fermentation processes for the oxidation of octane to 1‐octanol by Pseudomonas oleovorans, and we discuss the economics of these processes. In both systems the two‐liquid phase system consists of an apolar phase with hexadecene as the apolar carrier solvent into which n‐octane is dissolved, while the cells are present in the aqueous phase. In one system, multiple‐batch fermentations are followed by continuous processing of the product from the separated apolar phase. The second system is based on alkane oxidation by continuously growing cultures, again followed by continuous processing of the product. Fewer fermentors were required and a higher space‐time‐yield was possible for production of 1‐octanol in a continuous process. The overall performance of each of these two systems has been modeled with Aspen software. Investment and operating costs were estimated with input from equipment manufacturers and bulk‐material suppliers. Based on this study, the production cost of 1‐octanol is about 7 US$kg⁻¹ when produced in the fed‐batch process, and 8 US$kg⁻¹ when produced continuously. The comparison of upstream and downstream capital costs and production costs showed significantly higher upstream costs for the fed‐batch process and slightly higher upstream costs for continuous fermentation. The largest cost contribution was due to variable production costs, mainly resulting from media costs. The organisms used in these systems are P. putida alk+ recombinants which oxidize alkanes, but cannot oxidize the resulting alkanols further. Hence, such cells need a second carbon source, which in these systems is glucose. Although the continuous process is about 10% more expensive than the fed‐batch process, improvements to reduce overall cost can be achieved more easily for continuous than for fed‐batch fermentation by decreasing the dilution rate while maintaining near constant productivity. Improvements relevant to both processes can be achieved by increasing the biocatalyst performance, which results in improved overall efficiency, decreased capital investment, and hence, decreased production cost. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 84: 459–477, 1999.     

Techno-economic modeling and assessment of cultivated meat: Impact of production bioreactor scale

Increases in global meat demands cannot be sustainably met with current methods of livestock farming, which has a substantial impact on greenhouse gas emissions, land use, water consumption, and farm animal welfare. Cultivated meat is a rapidly advancing technology that produces meat products by proliferating and differentiating animal stem cells in large bioreactors, avoiding conventional live-animal farming. While many companies are working in this area, there is a lack of existing infrastructure and experience at commercial scale, resulting in many technical bottlenecks such as scale-up of cell culture and media availability and costs. In this study, we evaluate theoretical cultivated beef production facilities with the goal of envisioning an industry with multiple facilities to produce in total 100,000,000 kg of cultured beef per year or ~0.14% of the annual global beef production. Using the computer-aided process design software, SuperPro Designer®, facilities are modeled to create a comprehensive analysis to highlight improvements that can lower the cost of such a production system and allow cultivated meat products to be competitive. Three facility scenarios are presented with different sized production reactors; ~42,000 L stirred tank bioreactor (STR) with a base case cost of goods sold (COGS) of $35/kg, ~211,000 L STR with a COGS of $25/kg, and ~262,000 L airlift reactor (ALR) with a COGS of $17/kg. This study outlines how advances in scaled up bioreactors, alternative bioreactor designs, and decreased media costs are necessary for commercialization of cultured meat products.     

Vitronectin enhances adhesion force and t-PA production of weakly adherent 293 cells exposed to a shear stress

The effects of shear stress on the adhesion andproductivity of 293 cells were studied quantitativelyand compared with those of Vero and human liver cells.These cells were cultured in polystyrene dishes byusing shear stress exposing equipment. 50% of 293cells cultured in 2% FBS supplemented medium detachedfrom the dish after 29 h of exposure to a shear stressof 0.10 Pa. On the other hand, 90% of Vero and humanliver cells remained on the dish under the samecondition. Observations with scanning electronmicroscopy about cell adhesion plaques on the surfaceof the dish showed that the area covered withlamellipodia and the number of microspikes for 293cells were found to be less than those of the othercell lines. Several attachment factors, especiallyvitronectin, were found to enhance the number ofmicrospikes and the adhesion force of 293 cells.Almost 100% of 293 cells remained on thevitronectin-coated dish after 40 h under 0.10 Pa ofshear stress. A higher shear stress (greater than 0.10Pa) caused a decrease in tissue plasminogen activator(t-PA) productivity of 293 cells. But 0.03 Pastimulated the t-PA secretion on the non-coated dish.Vitronectin also enhanced the t-PA secretion evenunder 0.10 Pa. These results indicate that theadhesion force of 293 cells is obviously weaker thanthat of the other cell lines, and vitronectin enhancesthe adhesion force and the productivity of 293 cellsexposed to a shear stress.     

Direct coupling of expanded bed adsorption with a downstream purification step

In the course of developing a cost-effective, scaleable process for the purification of a recombinant protein from Chinese hamster ovary (CHO) suspension cell culture, we investigated direct capture of this molecule using expanded bed adsorption (EBA). EBA combines clarification, purification, and concentration of the product into a single step. The unclarified bioreactor material was directly applied to a STREAMLINE 25 column containing an affinity STREAMLINE adsorbent. This work focused on simplifying the EBA operations and minimizing the overall processing time by running the EBA column unidirectionally, eluting in the expanded bed mode, and coupling the EBA column directly with ion exchange or hydrophobic interaction chromatography. Unidirectional EBA was clearly a simpler unit operation and did not require the use of specialized equipment. The increase in the elution pool volume was insignificant, especially when the EBA column was eluted directly onto the downstream column. Scale-down was simple and could be automated. Coupling of unidirectional EBA with a downstream purification step reduced processing time, equipment requirements and cost.     

Process cost and facility considerations in the selection of primary cell culture clarification technology

The bioreactor volume delineating the selection of primary clarification technology is not always easily defined. Development of a commercial scale process for the manufacture of therapeutic proteins requires scale‐up from a few liters to thousands of liters. While the separation techniques used for protein purification are largely conserved across scales, the separation techniques for primary cell culture clarification vary with scale. Process models were developed to compare monoclonal antibody production costs using two cell culture clarification technologies. One process model was created for cell culture clarification by disc stack centrifugation with depth filtration. A second process model was created for clarification by multi‐stage depth filtration. Analyses were performed to examine the influence of bioreactor volume, product titer, depth filter capacity, and facility utilization on overall operating costs. At bioreactor volumes <1,000 L, clarification using multi‐stage depth filtration offers cost savings compared to clarification using centrifugation. For bioreactor volumes >5,000 L, clarification using centrifugation followed by depth filtration offers significant cost savings. For bioreactor volumes of ～2,000 L, clarification costs are similar between depth filtration and centrifugation. At this scale, factors including facility utilization, available capital, ease of process development, implementation timelines, and process performance characterization play an important role in clarification technology selection. In the case study presented, a multi‐product facility selected multi‐stage depth filtration for cell culture clarification at the 500 and 2,000 L scales of operation. Facility implementation timelines, process development activities, equipment commissioning and validation, scale‐up effects, and process robustness are examined. © 2013 The Authors. American Institute of Chemical Engineers Biotechnol. Prog., 29:1239–1245, 2013     

Evaluating the Return in Ecosystem Services from Investment in Public Land Acquisitions

We evaluate the return on investment (ROI) from public land conservation in the state of Minnesota, USA. We use a spatially-explicit modeling tool, the Integrated Valuation of Ecosystem Services and Tradeoffs (InVEST), to estimate how changes in land use and land cover (LULC), including public land acquisitions for conservation, influence the joint provision and value of multiple ecosystem services. We calculate the ROI of a public conservation acquisition as the ratio of the present value of ecosystem services generated by the conservation to the cost of the conservation. For the land scenarios analyzed, carbon sequestration services generated the greatest benefits followed by water quality improvements and recreation opportunities. We found ROI values ranged from 0.21 to 5.28 depending on assumptions about future land use change, service values, and discount rate. Our study suggests conservation is a good investment as long as investments are targeted to areas with low land costs and high service values.     

Decisional tool for cost of goods analysis of bioartificial liver devices for routine clinical use

Background aimsBioartificial liver devices (BALs) are categorized as advanced therapy medicinal products (ATMPs) with the potential to provide temporary liver support for liver failure patients. However, to meet commercial demands, next-generation BAL manufacturing processes need to be designed that are scalable and financially feasible. The authors describe the development and application of a process economics decisional tool to determine the cost of goods (COG) of alternative BAL process flowsheets across a range of industrial scales.MethodsThe decisional tool comprised an information database linked to a process economics engine, with equipment sizing, resource consumption, capital investment and COG calculations for the whole bioprocess, from cell expansion and encapsulation to fluidized bed bioreactor (FBB) culture to cryopreservation and cryorecovery. Four different flowsheet configurations were evaluated across demands, with cell factories or microcarriers in suspension culture for the cell expansion step and single-use or stainless steel technology for the FBB culture step.ResultsThe tool outputs demonstrated that the lowest COG was achieved with microcarriers and stainless steel technology independent of the annual demand (1500–30 000 BALs/year). The analysis identified the key cost drivers were parameters impacting the medium volume and cost.ConclusionsThe tool outputs can be used to identify cost-effective and scalable bioprocesses early in the development process and minimize the risk of failing to meet commercial demands due to technology choices. The tool predictions serve as a useful benchmark for manufacturing ATMPs.     

Application of a 22L scale membrane bioreactor and cross-flow ultrafiltration to obtain purified chondroitin

Recently, the possibility of producing fructosylated chondroitin from the capsular polysaccharide of Escherichia coli O5:K4:H4, in fed‐batch and microfiltration experiments was assessed on a 2 L bioreactor. In this work, a first scale‐up step was set on a 22 L membrane reactor with modified baffles to insert ad hoc designed microfiltration modules permanently inside the bioreactor vessel. Moreover, the downstream polysaccharide purification process, recently established on the A¨?KTA cross‐flow instrument, was translated to a UNIFLUX‐10, a tangential flow filtration system suitable for prepilot scale. In particular, the microfiltered permeates obtained throughout the fermentation, and the supernatant recovered from the centrifuged broth at the end of the process, were treated as two separate samples in the following ultrafiltration procedure, and the differences in the two streams and how these affected the ultrafiltration/diafiltration process performance were analysed. The total amount of K4 capsular polysaccharide was about 85% in the broth and 15% in the microfiltered permeates. However, the downstream treatment was more efficient when applied to the latter. The major contaminant, the lipopolysaccharide, could easily be separated by a mild hydrolysis that also results in the elimination of the unwanted fructosyl residue, which is linked to the C‐3 of glucuronic acid residues. The tangential ultrafiltration/diafiltration protocols developed in a previous work were effectively scaled‐up, and therefore in this research proof of principle was established for the biotechnological production of chondroitin from the wild‐type strain E. coli O5:K4:H4. The complete downstream procedure yielded about 80% chondroitin with 90% purity. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 28: 1012–1018, 2012     

马铃薯密码子用法分析及其在t-PA基因密码子改造上的应用

采用bioperl-1.0 工具在红旗LINUX系统下自编了密码子分析软件;通过对马铃薯98个蛋白质编码基因序列（codon DNA sequence）的分析,计算出了马铃薯的密码子用法,并确定出了马铃薯的4个高表达优越密码子;依据马铃薯密码子用法和高表达优越密码子分析结果,对t-PA基因序列进行了密码子的改造,得到了具有马铃薯密码子使用特点的t-PA基因序列,从而为以马铃薯为生物反应器高效生产t-PA奠定了分子基础。 Abstract:Bioperl-1.0 was used under Hongqi LINUX system to programm the codon analysis software.According to the analysis of 98 codon DNA sequences with this software,the codon usage in potato was calculated and 4 codons have been inferred to the optimal codons.The codons of tissue plasminogen activator (t-PA) gene sequence have been reconstructed according to the results.The t-PA gene sequence containing the optimal codons of potato will be used for t-PA production by potato bioreactor.     

Application of bioreactors for large-scale micropropagation systems of plants

Summary The application of bioreactor culture techniques for plant micropropagation is regarded as one of the ways to reduce production cost by scaling-up and automation. Recent experiments are restricted to a small number of species that, however, demonstrate the feasibility of this technology. Periodic immersion liquid culture using ebb and flood system and column-type bubble bioreactors equipped with a raft support system to maintain plant tissues at the air and liquid interface were found to be suitable for micropropagation of plants via the organogenic pathway. Balloon-type bubble bioreactors proved to be fit for micropropagation via somatic embryogenesis with less shear stress on cultured cells. Several cultivars of Lilium were successfully propagated using a two-stage culture method in one bioreactor. A large number of small-scale segments were cultured for 4 wk with periodic immersion liquid culture to induce multiple bulblets from each segment, then the bulblet induction medium was changed into bulblet growth medium by employing a submerged liquid bioreactor system. This culture method resulted in a nearly 10-fold increase in bulblet growth compared to conventional culture with solid medium. About 20 000 cuttings of virus-free potato could be obtained from 120 singlenode explants in a 20-liter balloon-type bubble bioreactor after 8 wk of culture. The percentage of ex vitro survival and root induction of the cuttings was more than 95%. Other successful results were obtained from the micropropagation and transplant production of chrysanthemum, sweetpotato, Chinese foxglove. Propagation systems via somatic embryogenesis in Acanthopanax koreanum and thornless Aralia elata were established using a liquid suspension of embryogenic determined cells. More than 500 000 somatic embryos in different stages were harvested from a 10-liter balloon-type bubble bioreactor after a 6-wk culture. Further development of these embryos in solid medium and eventually in the field was successful. The bioreactor system could reduce initial and operational cost for micropropagation, but further development of sophisticated technology might be needed to apply this system to plant micropropagation industries.