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BMC Proceedings 2013, Volume 7 Suppl 6 http://www.biomedcentral.com/bmcproc/supplements/7/S6 Page 58 of 151 Figure 1(abstract P38) Antibody titer measured by Biacore in SF fed-batch process (g/L). (A) 31 selected Rebmab100 clones measured on the last day of a 50 mL SF fed-batch culture. (B) Maximum (grey bars) and 2 weeks (black bars) mAb productivity obtained in a 200 mL SF fed-batch culture for the 7 stable Rebmab100 clones. The number above the grey bars indicates the day when maximum mAb productivity occurred. picking clones that would not grow isolated in LDC. Both CP-FL and LDC procedures proved efficient for generating high productive and stable cell clones. Overall productivity for individual clones depends on specific productivity, cell density and viability along time, allowing accumulation of the antibody. CP-FL clones reached maximum productivity at an earlier stage (2 weeks) of the 200 mL SF fed-batch experiment, which represents an advantage during the manufacturing process. The 4 lead clones will be submitted to bioreactor runs to evaluate the most suitable clone for the Rebmab100 mAb to be used in clinical trials and eventually to go under production. Acknowledgements: We acknowledge the excellent technical support of Denis N Aranha and José M Oliveira. WearegratefultoDr.MariaTA Rodrigues for logistics support. This work was supported by FAPESP, FINEP, CNPq, Fundação Butantan, and Recepta-biopharma. References 1. Browne SM, Al-Rubeai M: Selection methods for high-producing mammalian cell lines. Trends Biotechnol 2007, 25:425-432. 2. Kitamura K, Stockert E, Garin-Chesa P, Welt S, Lloyd KO, Armour KL, Wallace TP, Harris WJ, Carr FJ, Old LJ: Specificity analysis of blood group Lewis-y (Le(y)) antibodies generated against synthetic and natural Le(y) determinants. Proc Natl Acad Sci USA 1994, 91:12957-12961. 3. Scott AM, Geleick D, Rubira M, Clarke K, Nice EC, Smyth FE, Richards EC, Carr FJ, Harris WJ, Armour KL, Rood J. Kypridis A, Kronina V, Murphy R, Lee FT, Liu Z, Kitamura K, Ritter G, Laughton K, Hoffman E, Burgess AW, Old LJ: Construction, production, and characterization of humanized anti-Lewis Y monoclonal antibody 3S193 for targeted immunotherapy of solid tumors. Cancer Res 2000, 60:3254-3261. 4. Kelly MP, Lee FT, Smyth FE, Brechbiel MW, Scott AM: Enhanced efficacy of 90Y-radiolabeled anti-Lewis Y humanized monoclonal antibody hu3S193 and paclitaxel combined-modality radioimmunotherapy in a breast cancer model. J Nucl Med 2006, 47:716-725. 5. Scott AM, Tebbutt N, Lee FT, Cavicchiolo T, Liu Z, Gill S, Poon AM, Hopkins W, Smyth FE, Murone G, MacGregor D, Papenfuss AT, Chappell B, Saunder TH, Brechbiel MW, Davis ID, Murphy R, Chong G, Hoffman EW, Old LJ: A phase I biodistribution and pharmacokinetic trial of humanized monoclonal antibody Hu3s193 in patients with advanced epithelial cancers that express the Lewis-Y antigen. Clin Cancer Res 2007, 13:3286-3292. 6. Smaletz O, Diz MPD, Carmo CC, Sabbaga J, Cunha GF, Azevedo SJ, Maluf FC, Barrios CH, Costa RL, Fontana AG, Alves VA, Moro AM, Scott EW, Hoffman EW, Old LJ: Anti-LeY monoclonal antibody (mAb) hu3S193 (Rebmab100) in patients with advanced platinum resistant/refractory (PRR) ovarian cancer (OC), primary peritoneal cancer (PPC), or fallopian tube cancer (FTC). ASCO Annual Meeting, 2011, Chicago. J Clin Oncol 2011, 29:5078. P39 Impact of single-use technology on continuous bioprocessing William G Whitford * , Brandon L Pence Thermo Fisher Scientific, 925 West 1800 South, Logan, Utah 84321, USA E-mail: bill.whitford@thermofisher.com BMC Proceedings 2013, 7(Suppl 6):P39 Background: Single-use (SU) technologies supply a number of values to any mode of bioprocessing, but can provide some specific and enabling features in continuous bioprocessing (CB) implementations [1-3]. Most every operation in a CB process train is now supported by a commercially available single-use, or at least hybrid, solution (Figure 1). First of all, many of the SU equipment and solutions being developed for batch bioproduction have the same or related application in CB systems. Examples here include simple equipment such as tubings and connectors, to more complex applications such as the cryopreservation of large working stock aliquots in flexible bioprocess containers (BPCs). The list of CB-supporting SU technologies being developed is large and growing. Results: A SU advantage in process development is its supports of an open architecture approach and a number of hybrid designs. Such designs include combining reusable and single-use systems, or between divergent suppliers of particular equipment. Especially in bioproduction, the many flexibilities of SU support a manufacturing platform of exceptional efficiency, adaptability, and operational ease. Advances designs in SU transfer tubing, manifold design and container porting also supports creativity in process design. This is of particular value in designing a process with such demands as entirely new flow paths or lot designations, such for CB. SU systems upstream provide a reduced footprint and eliminate of the need for cleaning and sterilization service. This complements perfusion culture’s inherently smaller size and independence from cleaning for extended periods of time.
BMC Proceedings 2013, Volume 7 Suppl 6 http://www.biomedcentral.com/bmcproc/supplements/7/S6 Page 59 of 151 Figure 1(abstract P39) Hybrid intensified perfusion-based continuous bioproduction in a Thermo Scientific HyPerforma S.U.B. TK 250L supported by yhe Refine Technology ATF System. Several newer approaches to formulating process fluids support the concept of CB. Single-use mixing systems are typically constructed of a rigid containment system with a motor and controls driving radiation-sterilized single-use bags equipped with disposable impeller assemblies. From a variety of manufacturers there are a number of distinct approaches to motor/disposable impeller assembly linkages, tubing lines and connections. Also appearing are a number of exciting SU sampling, sensing, and monitoring solutions. Single-use powder containers permit seamless transfer between powder and liquid formulation steps, and the ridged mixing containers are available in jacketed stainless steel for heating and cooling requirements. Surprisingly, the “topping-up” of large-scale single-use fluid containers with newly prepared buffer to provide a virtually unlimited and constant supply of each buffer/media type can be validated for GMP manufacturing procedures. Process flexibility is a key feature in both SU and CB. CB contributes to overall process flexibility in that equipment tends to be easy to clean, inspect and maintain − and generally promotes simple and rapid product changeover. SU systems can provide similar flexibility and ease product changeover because they tend to be more modular and transportable than much of the older batch equipment. In fact the size, configuration and reduced service requirements of SU systems actually encourage diversity of physical location within a suite or plant, as well as re-location to other manufacturing sites. Due to its inherent demand for immediate process data and control capabilities, CB supports initiatives in continuous quality verification (CQV), continuous process verification (CPV), and real-time release (RTR). Although CB will not be feasible for all products and processes, many implementations well-support a “platform” approach, in which a single process supports more than one product. CB most always shortens the process stream, reduces downtime, and greatly reduces handling of intermediates. These features complement the operational efficiencies of SU systems, contributing to a greatly reduced cumulative processing time for the API. Furthermore, they greatly simplify production trains and inherently facilitate application of closed processing approaches to individual operations and even processes. Especially in bioproduction, the modularity and integral gamma irradiation sterility of SU combined with the sustained operation of CB promise the appearance of platforms of unparalleled operational simplicity and convenience. The heart of a CB approach is the bioreactor. Perfusion bioreactors have been successfully employed in bioproduction, even biopharmaceutical production, for decades. And, rather remarkably, disposable bioreactors have been available for nearly 20 years. At the research scale there have even been single-use hollow fiber perfusion bioreactors available from a variety of vendors for over 40 years. However, only recently have commercially available SU and hybrid production-scale perfusion-capable equipment become available. The production-scale CB enabling SU bioreactor technologies now becoming commercial available include single-use and hybrid perfusioncapable reactors (Figure 1); a growing variety of SU and hybrid monitoring probes and sensors; SU pumps and fluid delivery automation of various design; and automated SU online sampling, interface, valving and feeding technologies. Their coordinated implementation in actual production settings with appropriate control is now beginning. Justified or not, concerns in the implementation of CB include performance reliability (incidence of failure), validation complexity, process control and economic justification. But for many processes, such previous limitations – or their perception – are being alleviated by advances in CB processing technology and OpEx driven advances bioprocess understanding, reactor monitoring and feedback control. However, while some CB attributes inherently provide immediate advantages (such as reduces reactor
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