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Integration of Aqueous Two-Phase Extraction As Cell Harvest and Capture Operation in the Manufacturing Process of Monoclonal Antibodies

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Integration of Aqueous Two-Phase Extraction As Cell Harvest and Capture Operation in the Manufacturing Process of Monoclonal Antibodies antibodies Article Integration of Aqueous Two-Phase Extraction as Cell Harvest and Capture Operation in the Manufacturing Process of Monoclonal Antibodies Axel Schmidt 1, Michael Richter 2, Frederik Rudolph 2 and Jochen Strube 1,* 1 Institute for Separation and Process Technology, Clausthal University of Technology, Leibnizstraße 15, 38678 Clausthal-Zellerfeld, Germany; [email protected] 2 Boehringer Ingelheim Pharma GmbH & Co. KG, Bioprocess + Pharma. Dev. Biologicals, Birkendorfer Strasse 65, 88397 Biberach an der Riss, Germany; [email protected] (M.R.); [email protected] (F.R.) * Correspondence: [email protected]; Tel.: +49-5323-72-2200 Received: 30 October 2017; Accepted: 20 November 2017; Published: 1 December 2017 Abstract: Substantial improvements have been made to cell culturing processes (e.g., higher product titer) in recent years by raising cell densities and optimizing cultivation time. However, this has been accompanied by an increase in product-related impurities and therefore greater challenges in subsequent clarification and capture operations. Considering the paradigm shift towards the design of continuously operating dedicated plants at smaller scales—with or without disposable technology—for treating smaller patient populations due to new indications or personalized medicine approaches, the rising need for new, innovative strategies for both clarification and capture technology becomes evident. Aqueous two-phase extraction (ATPE) is now considered to be a feasible unit operation, e.g., for the capture of monoclonal antibodies or recombinant proteins. However, most of the published work so far investigates the applicability of ATPE in antibody-manufacturing processes at the lab-scale and for the most part, only during the capture step. This work shows the integration of ATPE as a combined harvest and capture step into a downstream process. Additionally, a model is applied that allows early prediction of settler dimensions with high prediction accuracy. Finally, a reliable process development concept, which guides through the necessary steps, starting from the definition of the separation task to the final stages of integration and scale-up, is presented. Keywords: interfacial partitioning; cell harvest; capture; aqueous two-phase extraction; horizontal settler; static mixer; process integration; continuous phase separation 1. Introduction Clarification processes and their corresponding devices, which are already well established in other industry sectors, e.g., flocculation, precipitation, or flotation, are increasingly being taken into consideration as alternatives in monoclonal antibodies (mAb) production in spite of individual limitations like process robustness, process cost, toxicity of flocculation or precipitation agents, and easy scale-up [1]. A quite promising concept is the application of aqueous two-phase extraction (ATPE) as a combined harvest and capture step, especially since this approach deals with all the aforementioned issues. Interfacial partitioning of cells, cell debris, and other bioparticles occurs when a mixed system composed of phase-forming components begins to separate into its specific light and heavy phases [2–4]. During the process of settling and coalescence, particles and small solid objects accumulate at the surface of the dispersed phase (Figure1). This phenomenon can be explained by the fact that the partitioning behavior of small particles is strongly dependent on surface forces [5]. Antibodies 2017, 6, 21; doi:10.3390/antib6040021 www.mdpi.com/journal/antibodies Antibodies 2017, 6, 21 2 of 19 the surface of the dispersed phase (Figure 1). This phenomenon can be explained by the fact that the Antibodiespartitioning2017, 6behavior, 21 of small particles is strongly dependent on surface forces [5]. 2 of 19 FigureFigure 1.1. Cell clearance through interfacialinterfacial partitioning.partitioning. ATPE:ATPE: AqueousAqueous two-phasetwo-phase extraction.extraction. ApproachesApproaches forfor thethe combinationcombination ofof liquid–liquidliquid–liquid extractionextraction (LLE)(LLE) andand thethe separationseparation ofof smallsmall particlesparticles byby adsorptionadsorption towardstowards aa disperseddispersed phasephase areare wellwell investigated.investigated. TheseThese includeinclude interfacialinterfacial partitioningpartitioning forfor the the recovery recovery of bioparticles of bioparticles in general, in general, three-phase three partitioning-phase partition (TPP)ing where (TPP) interfacial where partitioninginterfacial partitioning is combined is combined with the precipitationwith the precipitation of proteins, of proteins or more, or recently, more recently aqueous, aqueous two-phase two- flotation,phase flotation, where gaswhere bubbles gas bubbles are introduced are introduced as a dispersed as a dispersed third phase third into phase an already into an separated already aqueousseparated two-phase aqueous systemtwo-phase (ATPS) system [6–8 ].(ATPS) However, [6–8]. most However, approaches most so approaches far design the so processfar design around the theprocess particle-loaded around the phase particle because-loaded the solidphase phase because contains the solid the product. phase contains Some research the product. has also Some been conductedresearch has towards also been interfacial conducted partitioning towards for interfacial the removal partitioning of unwanted for particles the removal from aof feed unwanted stream, whichparticles is especially from a feed necessary stream, during which the is clarification especially ofnecessary cultivation during broths the in clarification the manufacturing of cultivation process ofbroths monoclonal in the manufacturing antibodies. However, process toof establishmonoclonal this antibodies. technology However, in industry, to establish there is also this the technology need for ain process industry, development there is also strategy the thatneed guides for a throughprocess thedevelopment necessary steps, strategy starting that fromguides the definitionthrough the of thenecessary exact separation steps, starting task, from to the the experiments definition of necessary the exact separation for the determination task, to the experiments of crucial process necessary and modelfor the parameters,determination up of to thecrucial design process considerations and model for parameters, optimal equipment up to the dimensions.design considerations In this work, for anoptimal ATPE equipment process is outlined dimensions. for the In clarification this work, an of upATPE to 12,000 process L ofis cultivationoutlined for broth the clarification in a time window of up ofto less12,000 than L of 3 h.cultivation broth in a time window of less than 3 h. 2.2. TheoryTheory 2.1.2.1. GeneralGeneral ConsiderationsConsiderations UnlikeUnlike inin thethe conventionalconventional productionproduction processprocess ofof mAb,mAb, centrifugationcentrifugation andand microfiltrationmicrofiltration asas harvestharvest andand clearance clearance operations operations are are replaced replaced with with the outlinedthe outlined ATPE ATPE process. process. After After the filtration the filtration trains, thetrains, product the product stream can, stream depending can, depending on the future on the process future strategy, process be strategy, further purified be further by precipitation purified by orprecipitation by integrated or counter-currentby integrated chromatographycounter-current (iCCC)chroma fortography continuous (iCCC) capture, for continuous replacing traditional capture, proteinreplacing A chromatography,traditional protein as wells A chromatography, as ion-exchange and as hydrophobicwells as ion interaction-exchange chromatography and hydrophobic for furtherinteraction purification chromatography and polishing for further [9–14]. purification and polishing [9–14]. TheThe flowsheetflowsheet ofof thethe discusseddiscussed process,process, including the two purificationpurification and polishing operations,operations, isis shownshown inin FigureFigure2 2.. It It provides provides a a precise precise and and straightforward straightforward view view of of how how ATPE ATPE is is integrated integrated into into thethe overalloverall purificationpurification strategy.strategy. ToTo quicklyquickly getget anan overviewoverview ofof allall process-relevantprocess-relevant effects,effects, itit isis helpfulhelpful toto constructconstruct aa cause-and-effectcause-and-effect diagram,diagram, alsoalso knownknown asas anan IshikawaIshikawa oror fishfish bonebone diagram.diagram. ItIt isis meantmeant to illustrate the different possible sources of reduced or insufficient process performance. The diagram Antibodies 2017, 6, 21 3 of 19 AntibodiesAntibodies2017 2017, 6, ,6 21, 21 3 3of of 19 19 to illustrate the different possible sources of reduced or insufficient process performance. The to illustrate the different possible sources of reduced or insufficient process performance. The diagram is constructed of primary branches, which organize specific groups of effects. They can be isdiagram constructed is constructed of primary of branches,primary branches, which organize which organize specific specific groups ofgroups effects. of effects. They can They be can fanned be fanned out further into major branches, representing major causes. Minor branches can be integrated outfanned further out intofurther major into branches, major branches, representing representing major major causes. causes.
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