DOCSLIB.ORG

Aqueous Two-Phase Extraction of Proteins and Enzymes Using Tetraalkylammonium-Based Ionic Liquids

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Aqueous Two-Phase Extraction of Proteins and Enzymes Using Tetraalkylammonium-Based Ionic Liquids Aqueous two-phase extraction of proteins and enzymes using tetraalkylammonium-based ionic liquids Dissertation zur Erlangung des akademischen Grades doctor rerum naturalium (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Universität Rostock vorgelegt von Susanne Elisabeth Dreyer Rostock 2008 urn:nbn:de:gbv:28-diss2009-0038-8 Referent: Prof. Dr. Udo Kragl, Universität Rostock Koreferenten: Prof. Dr. Uwe Bornscheuer, Universität Greifswald Prof. Dr. Jürgen Hubbuch, Universität Karlsruhe Tag der Verteidigung: 27. Januar 2009 Die vorliegende Arbeit wurde im Zeitraum von Juli 2005 bis Oktober 2008 am Institut für Chemie der Universität Rostock angefertigt. Danke Mein großer Dank gilt Herrn Prof. Dr. Udo Kragl, der es mir durch eine interessante Themenstellung nicht nur ermöglicht hat, in der Arbeitsgruppe „Technische Chemie“ zu promovieren, sondern mir durch viele Anregungen und Diskussionen auch durch die Höhen und Tiefen der Arbeit geholfen hat. Weiterhin möchte ich mich für die zahlreichen Möglichkeiten bedanken, an Konferenzen und Tagungen im In- und Ausland teilzunehmen, die oft mit interessanten neuen Ideen und Erfahrungen verbunden waren. Weiterhin möchte ich Herrn Prof. Dr. Uwe Bornscheuer und Herrn Prof. Dr. Jürgen Hubbuch für die freundliche Übernahme des Koreferates dieser Arbeit danken. Mein Dank geht auch an Dr. Thomas Daußmann für die Bereitstellung der Alkohol- dehydrogenase aus Lactobacillus brevis und der T ADH, sowie an die Firma Solvent Innovation GmbH für interessante Anregungen, viele Informationen und die Proben der ionischen Flüssigkeiten. Dr. Thomas Schmidt danke ich für die Leihgabe des Viskosimeters und die Einführung in dessen Bedienung. Ein großer Dank geht insbesondere an Herrn Dr. Ruth, Katja Neubauer und Christina Bathke, die mir in analytischen Fragen große Hilfestellungen und gute Ratschläge gegeben haben. Sebastian Trapp, Christian Westendorf, Juliana Ratzka, Florian Stein und Paulina Salim danke ich für die Unterstützung und interessanten Ergebnisse, die sie im Rahmen von ihren Tätigkeiten als studentische Hilfskraft bzw. wissenschaftliche Mitarbeiter zu dieser Arbeit beigetragen haben. Weiterhin möchte ich der Degussa Stiftung sowohl für die finanzielle Unterstützung als auch die Einladungen zu den Stipendiaten-Treffen danken, die nicht nur interessant und informativ, sondern stets auch persönlich bereichernd waren. Dem Forschungsschwerpunkt SPP1191- Ionic Liquids danke ich ebenfalls für die finanzielle Unterstützung. Danke, danke, danke! an die ganze Arbeitsgruppe „Technische Chemie”, die mich so herzlich in ihren Kreis aufgenommen und geduldig meine rheinländischen Eigenarten ertragen hat. Danke für die schöne, lustige und manchmal chaotische Arbeitsatmosphäre, die gemütlichen Tee- und Kuchenpausen, die Kicker-Spiele und die vielen Biologen-Witze! Besonders möchte ich mich noch einmal bei Julia, Katja, Daniela, Sandra, Vera, Annett und Jan bedanken, die mir nicht nur bei wissenschaftlichen, sondern auch persönlichen Fragen und Problemen immer zur Seite standen! Ein ganz großes „Danke“ auch an Frau Freitag, Frau Plagemann und Herrn Spallek für viele nette und aufmunternde Gespräche, Hilfestellung in allen Lebenslagen und die Leihgabe einer Latzhose. Mein besonderer Dank geht an meine Großeltern, meine Eltern Eva und Hinnerk Dreyer, meine Schwestern Barbara und Pisa, und an Staffan. Danke für die Unterstützung und Hilfe in jeder Situation und Lebenslage. Danke, dass Ihr trotz der großen Entfernung immer da ward! Für Eva, Hinnerk, Barbara und Pisa De Möh von hück es de joot ahl Zick vun üvvermorje. Our troubles of today are those ‘good old days’ of tomorrow. Kölsche Weisheit Abstract To meet the challenges of a growing market of industrial biotechnology, the development of new and innovative downstream methods for proteins and enzymes is of great importance. Within the scope of this thesis, the application of ionic liquids for the generation of aqueous two-phase systems and their potential use as an alternative technique for the purification of catalytically active enzymes was evaluated. The first objective comprised the characterisation of ionic liquid-based aqueous two-phase systems (IL-based ATPS). Several factors influencing the phase formation and composition were investigated, such as the type of phase-forming compounds, temperature and pH. By studying the partitioning of different model proteins, correlations between the protein features and system properties could be found, thereby providing a deeper understanding of the mechanisms influencing the partition process. Based on these findings, electrostatic interaction has been identified as the main driving force of protein partitioning in IL-based ATPS. Moreover, a statistical model based on the model proteins’ physico-chemical properties has been established and provides a good start for predicting the protein partitioning in IL-based ATPS. With regard to the purification of enzymes, the main obstacle in developing a suitable extraction method represents the maintenance of the stability and the catalytic activity of the biocatalyst. Therefore, the application of IL-based ATPS for biocompatible extraction of enzymes from complex mixtures such as cell lysates was investigated. It was demonstrated that the ionic liquid AmmoengTM 110 in combination with an inorganic salt (K2HPO4/KH2PO4) can be used to establish an IL-based ATPS which provides the necessary conditions of pH and biocompatibility in order to maintain enzyme activity. Using efficient experimental design the partitioning of an ADH from Lactobacillus brevis (LB ADH) and an ADH from a thermophilic organism (T ADH) from a cell extract of E. coli was optimised with respect to the enrichment of the enzyme in the IL-containing phase. The specific enzyme activity in the upper phase could be increased to 206.7 ± 5.3 % for LB ADH and 403.4 ± 5.4 % for T ADH. The presence of ionic liquid within IL-based ATPS was also found to be advantageous thanks to its stability enhancing effect on both enzymes and its solubility enhancing effect on hydrophobic substrates. Thus, it could be shown that the IL-based ATPS offers the opportunity to combine the purification process of active biocatalysts with the performance of enzyme-catalysed reactions especially in case of the conversion of hydrophobic substrates. Advantages such as improved conversion and yield could be observed. Parts of this thesis as well as further results not included in this work have already been presented, published and filed as a patent. Publications Dreyer S, Lembrecht J, Schumacher J, Kragl U. (2007). Enzyme catalysis in non-aqueous media past-present-future. In R. Patel (Ed.) Biocatalysis in the Pharmaceutical and Biotechnology Industries, Taylor & Francis Group, Chapter 33, 791-829 Klembt S, Dreyer S, Eckstein M, Kragl U. (2007). Biocatalytic reactions in ionic liquids. In Wasserscheid, P., Welton, T. (eds.): Ionic Liquids in Synthesis (2nd edition), Wiley-VCH Weinheim Dreyer S, Kragl U. (2008). Ionic liquids for aqueous two phase extraction and stabilization of enzymes. Biotechnology and Bioengineering, Vol. 99, 6, 1416-1424 Dreyer S, Salim P, Kragl U. (2008). Driving forces of protein partitioning in an ionic liquid- based aqueous two phase system. Submitted to Biochemical Engineering Journal Patents Dreyer S, Kragl U. (2007). Verfahren zur Extraktion von Biomolekülen (Process for the extraction of biomolecules). German patent application, 102007001347.9, 04/01/2007 Altrichter J, Kragl U, Dreyer S (2008). Stabilisierung von Zellen durch ionische Flüssigkeiten (Stabilisation of cells by ionic liquids). German patent application, 102008039734.2, 26/08/2008 Oral presentations Dreyer S. (19/06/2007). Application of ionic liquids for aqueous two phase extraction. International Conference on Biopartitioning and Purification, Lisbon Dreyer S. (16/11/2007). Ionic liquids for the purification and stabilisation of proteins. Workshop Interaction of Ionic Liquids and Proteins, Rostock Dreyer S. (21/02/2008). Use of ionic liquids for protein purification. SPP1191 Ionic Liquid Winterschool, Leipzig Dreyer S. (03/09/2008). Ionic liquids for aqueous two phase extraction of enzymes: application and understanding. Presented by Julia Duwensee, Biocat, Hamburg Poster presentations Dreyer S, Trapp S, Kragl U. (30/05 01/06/2007). Stabilisation of two different alcohol dehydrogenases by a readily available ionic liquid. European Bioperspectives, Köln Dreyer S, Kragl U. (05/08 10/08/2007). Ionic liquid-based aqueous two phase extraction of enzymes. 2nd International Congress on Ionic Liquids (COIL 2), Yokohama Dreyer S, Kragl U. (09/10 - 11/10/2007). Ionische Flüssigkeiten für die Extraktion und Stabilisierung von Proteinen. Biotechnica, Hannover Dreyer S, Kragl U. (15/11 16/11/2007). Ionic liquid-based aqueous two phase extraction of enzymes. Workshop Interaction of Ionic Liquids and Proteins, Rostock Dreyer S, Kragl U. (27/03 - 29/03/2008). Protein distribution in ionic liquid-based aqueous two phase systems. International 10th Frühjahrssymposium of GDCh JCF, Rostock Index I I Index 1 Introduction .......................................................................................................1 2 Scope of work....................................................................................................4 3 Literature review ...............................................................................................7 3.1 Purification of proteins and enzymes ........................................................................7
Load more

Recommended publications
  • Protocols and Tips in Protein Purification
    Department of Molecular Biology & Biotechnology Protocols and tips in protein purification or How to purify protein in one day Second edition 2018 2 Contents I. Introduction 7 II. General sequence of protein purification procedures 9 Preparation of equipment and reagents 9 Preparation and use of stock solutions 10 Chromatography system 11 Preparation of chromatographic columns 13 Preparation of crude extract (cell free extract or soluble proteins fraction) 17 Pre chromatographic steps 18 Chromatographic steps 18 Sequence of operations during IEC and HIC 18 Ion exchange chromatography (IEC) 19 Hydrophobic interaction chromatography (HIC) 21 Gel filtration (SEC) 22 Affinity chromatography 24 Purification of His-tagged proteins 25 Purification of GST-tagged proteins 26 Purification of MBP-tagged proteins 26 Low affinity chromatography 26 III. “Common sense” strategy in protein purification 27 General principles and tips in “common sense” strategy 27 Algorithm for development of purification protocol for soluble over expressed protein 29 Brief scheme of purification of soluble protein 36 Timing for refined purification protocol of soluble over -expressed protein 37 DNA-binding proteins 38 IV. Protocols 41 1. Preparation of the stock solutions 41 2. Quick and effective cell disruption and preparation of the cell free extract 42 3. Protamin sulphate (PS) treatment 43 4. Analytical ammonium sulphate cut (AM cut) 43 5. Preparative ammonium sulphate cut 43 6. Precipitation of proteins by ammonium sulphate 44 7. Recovery of protein from the ammonium sulphate precipitate 44 8. Analysis of solubility of expression 45 9. Analysis of expression for low expressed His tagged protein 46 10. Bio-Rad protein assay Sveta’s easy protocol 47 11.
    [Show full text]
  • Anatomy and Physiology of Peritoneal Dialysis
    Anatomy and Physiology of Peritoneal Dialysis Isaac Teitelbaum, MD Professor of Medicine Director, Acute & Home Dialysis Programs University of Colorado Hospital Denver, Colorado •1 Outline • Peritoneal cavity as a dialysis system • Models of peritoneal transport • Physiology of peritoneal transport Inverse relationship between solute transport and ultrafiltration • Kinetics of peritoneal transport • Synthesis & Application • Middle Molecules Anatomy of The Peritoneum • The lining of the abdominal cavity • Two layers: parietal - lines the anterior wall and undersurface of the diaphragm - 20% of total SA; blood supply from abdominal wall visceral - covers the abdominal organs - 80% of total SA; blood supply from mesenteric aa and portal vv Gokal R, Textbook of PD, pp. 61-70 •3 Anatomy of The Peritoneum • Size 1.5 – 2 m2; approximates BSA • Highly Vascular • Semi-permeable/bi-directional • “Lymphatic” drainage through diaphragmatic stomata • Continuous with Fallopian Tubes in females Gokal R, Textbook of PD, pp. 61-70 1. The two main properties of the peritoneal membrane are: a. Semi permeable – this allows substances of certain sizes to move from an area of greater concentration to less concentration. b. Bi Directional - substances move in either direction across the membrane. 2. So-called “lymphatic” drainage refers to bulk flow from the peritoneal cavity back to the circulation. This actually occurs across tissues as well as lymphatics. As this is convective flow, dissolved solutes move with the fluid. Thus, fluid reabsorption results in loss of solute clearance as well as loss of fluid removal. 3. It is important to be aware of the continuity of the peritoneal cavity with the Fallopian tubes as retrograde menstruation- which may occur in any woman but goes undetected- will cause bloody dialysate and create concern in the PD patient.
    [Show full text]
  • Water Treatment for Hemodialysis
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Hong Kong JournalJ Nephrol of 2001;3(1):7-14.Nephrology MKH TONG, et al 2001;3(1):7-14. REVIEW ARTICLE Water treatment for hemodialysis Matthew Ka-Hang TONG1, Wei WANG2, Tze-Hoi KWAN1, Lawrence CHAN2, Tak-Cheung AU1 1Department of Medicine, Tuen Mun Hospital, Tuen Mun, Hong Kong; 2Division of Renal Diseases and Hypertension, Department of Medicine, University of Colorado Health Sciences Center, Denver, Colorado, USA. Abstract Water treatment plays a vital role in the delivery of safe and effective hemodialysis (HD). Ensuring that water quality meets the American Association for the Advancement of Medical Instrumentation standards and recommendations (or equivalent) is necessary to reduce the incidence of chemical hazards and endotoxemia associated with the use of water for HD. This review will discuss the principles of water treatment for HD, the essential components of water purification, the recommended system monitoring and maintenance procedures, and some of the historical incidents of adverse reactions that resulted from the use of contaminated dialysis water. Key words: Dialysis solutions, Hemodialysis (HD), Pyrogenic reactions, Reverse osmosis (RO), Water purification ! !"#$%&'()*EeaF !"#$%&'()*+,-./0123456789%: ea !"#$%&'()*+,-*./0123456789ea !"#$%& !"#$%&'()#*+,-./0123456789:;<=> INTRODUCTION can build up to toxic levels, causing long-term physical Since the early 1960s, hemodialysis (HD) has been harm, and other substances are immediately toxic and increasingly used for the treatment of acute renal failure can cause death. A normal person also encounter these and end-stage renal failure. Technologic advances in contaminants through drinking water, but the healthy dialyzer membranes, dialysis machines and vascular kidney is able to remove these substances.
    [Show full text]
  • 1 Downstream Processing of Biotechnology Products
    1 1 Downstream Processing of Biotechnology Products 1.1 Introduction Biological products are important for many applications including biotransforma- tions, diagnostics, research and development, and in the food, pharmaceutical, and cosmetics industries. For certain applications, biological products can be used as crude extracts with little or no purifi cation. However, biopharmaceuticals typi- cally require exceptional purity, making downstream processing a critical compo- nent of the overall process. From the regulatory viewpoint, the production process itself defi nes the biopharmaceutical product rendering proper defi nition of effec- tive and effi cient downstream processing steps crucial early in process develop- ment. Currently, proteins are the most important biopharmaceuticals. The history of their development as industrial products goes back more than half a century. Blood plasma fractionation was the fi rst full - scale biopharmaceutical industry with a current annual production in the 100 - ton scale [1, 2] . Precipitation with organic solvents has been and continues to be the principal purifi cation tool in plasma fractionation, although, recently, chromatographic separation processes have also been integrated into this industry. Anti - venom antibodies and other anti - toxins extracted from animal sources are additional examples of early biopharmaceuti- cals, also purifi ed by a combination of precipitation, fi ltration and chromatogra- phy. In contrast, current biopharmaceuticals are almost exclusively produced by recombinant DNA technology. Chromatography and membrane fi ltration serve as the main tools for purifi cation for these products. Figure 1.1 shows the 2006 market share of various biopharmaceuticals. Approxi- mately one - third are antibodies or antibody fragments [3] , nearly 20% are erythro- poietins, and 14% are insulins.
    [Show full text]
  • UC Riverside UC Riverside Electronic Theses and Dissertations
    UC Riverside UC Riverside Electronic Theses and Dissertations Title Effect of Electrostatic Interactions on Performance of Electrically Conductive Ultrafiltration Membranes for Protein Fractionation Permalink https://escholarship.org/uc/item/79q015j9 Author Yeung, Raymond Kai-yen Publication Date 2017 License https://creativecommons.org/licenses/by/4.0/ 4.0 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA RIVERSIDE Effect of Electrostatic Interactions on Performance of Electrically Conductive Ultrafiltration Membranes for Protein Fractionation A Thesis submitted in partial satisfaction of the requirements for the degree of Master of Science in Bioengineering by Raymond Kai-yen Yeung September 2017 Thesis Committee: Dr. Victor G. J. Rodgers, Chairperson Dr. David Jassby Dr. Boris Hyle Park Copyright by Raymond Kai-yen Yeung 2017 The Thesis of Raymond Kai-yen Yeung is approved: Committee Chairperson University of California, Riverside ACKNOWLEDGEMENTS My sincerest gratitude goes to my advisor, Dr. Victor G. J. Rodgers, whose instruction and guidance have facilitated my development as a researcher and scholar. His advice, knowledge of the research area, and commitment to his students have been crucial in fostering my growth and challenging me to reach new heights. I deeply value the research opportunity and am grateful for his continuous support. I would like to thank the members of my thesis committee, Dr. David Jassby and Dr. Hyle Park, for their support and advice they have provided in my research and graduate studies. I would also like to thank Xiaobo (Ben) Zhu for patiently assisting me in the laboratory, Dr. Alexander Dudchenko for providing valuable feedback in our discussions, and Dr.
    [Show full text]
  • Aqueous Two-Phase System (ATPS): an Overview and Advances in Its
    Iqbal et al. Biological Procedures Online (2016) 18:18 DOI 10.1186/s12575-016-0048-8 REVIEW Open Access Aqueous two-phase system (ATPS): an overview and advances in its applications Mujahid Iqbal1, Yanfei Tao1*, Shuyu Xie1, Yufei Zhu1, Dongmei Chen1, Xu Wang1, Lingli Huang1, Dapeng Peng1, Adeel Sattar1, Muhammad Abu Bakr Shabbir2, Hafiz Iftikhar Hussain2, Saeed Ahmed2 and Zonghui Yuan1,2* Abstract Aqueous two-phase system (ATPS) is a liquid-liquid fractionation technique and has gained an interest because of great potential for the extraction, separation, purification and enrichment of proteins, membranes, viruses, enzymes, nucleic acids and other biomolecules both in industry and academia. Although, the partition behavior involved in the method is complex and difficult to predict. Current research shows that it has also been successfully used in the detection of veterinary drug residues in food, separation of precious metals, sewage treatment and a variety of other purposes. The ATPS is able to give high recovery yield and is easily to scale up. It is also very economic and environment friendly method. The aim of this review is to overview the basics of ATPS, optimization and its applications. Keywords: Aqueous two-phase system (ATPS), Biomolecule separation, Solvent extraction, Veterinary drug residues History and background hydrophobic groups [5] Interested readers about In 1896, Martinus Willem Beijerinck accidently found aqueous two-phase affinity partitioning (ATPAP) are the ATPS while mixing an aqueous solution of starch referred to an excellent review by Ruiz-Ruiz et al. [6]. and gelatin. However, its real application was discovered Water as the main component of both phases in ATPS by Per-Åke Albertsson.
    [Show full text]
  • Separation Techniques (Proteins)
    Separation techniques (Proteins) Desalting / Dialysis, Ultrafiltration Dialysis and Ultrafiltration Dialysis is an easy and efficient technique to separate biomolecules according to their molecular weight, but also usefull for small bioactive compounds release or binding studies. Applications include : Applications Area Biomolecule of interest ! desalting (removing salts) biochemistry (synthesis, proteins ! buffer exchange labeling, analysis) lipids ! fractionation production (purification,...) nucleic acids carbon hydrates ! drug binding studies pharmacology drugs, ligands, cytokines... Membranes 200 MWCO most molecules with MW >200 ! drug release in cell models or organisms 200 Da MWCO Dialysis is driven by a differential concentration gradient, discriminating biomolecules across a "semi-permeable" membrane. The membrane can be considered to display pores that retain molecules above a given size, and is permeable to small molecules. Membrane should be chosen with a Molecular Weight Cut-Off (MWCO) between the MW of molecules to recover in the sample compartment, and the MW of the molecules to remove (usually salts, by-products of reaction,...). See technical tip. ! MWCO is defined for globular proteins (90% of molecules with MW=MWCO are retained), in given conditions (buffer, temperature,...). So, the membrane choice may depend on the molecules shape, hydrophilicity,... (see the table for nucleic acids with CelluSep). For effective dialysis, the ratio (MW of molecules to retain/MW of salt to remove) passed should be superior to 25. ! Also, the dialysis rate and the selectivity of molecules discrimination are affected near the Proteomics MWCO. For quick desalting, the dialysis speed may be favored with MWCO far above MW of salts (closer to biomolecules), while for applications with small difference of MWs or with populations of dispersed molecules, the selectivity may be privileged by choosing a MWCO relatively close to small molecules to be removed.
    [Show full text]
  • What Can Cell Culture Flocculation Offer for Antibody Purification Processes
    Editorial 2 Editorial What can cell culture flocculation offer for antibody purification processes “The eventual goal of the application of flocculation in monoclonal antibody processing is to develop a separation unit operation alternative to chromatography, with equivalent separation efficiency, Pharm. Bioprocess. high process capacity, good facility fit and sound process economics.” Keywords: antibody • flocculation • harvest • impurity removal • purification Recent advances in cell culture processes solid content with wide particle distribution Yun (Kenneth) Kang have significantly increased monoclonal anti- and a high percentage of small particles fur- Author for correspondence: body (mAb) titers to levels as high as 30 g/l. ther complicate the harvest unit operation, BioProcess Sciences, Eli Lilly Not unexpectedly, these improvements have requiring significantly higher depth filter & Company, 450 East 29th Street, New York, NY 10016, USA resulted in a new set of starting conditions surface area which may exceed the existing [email protected] for downstream processing, including: ele- filtration train capacity. Harvest operations vated cell density levels (up to 108 cells/ml), have become extremely challenging, if not Dale L Ludwig BioProcess Sciences, Eli Lilly increased solid content (packed cell volume, impossible because of these issues. & Company, 450 East 29th Street, up to 40%) and increased soluble impurity Flocculation has been widely used for New York, NY 10016, USA levels such as host cell proteins (HCP) and many years in the chemical and food indus- Paul Balderes high molecular weight species (HMW; up tries, as well as in wastewater treatment. In BioProcess Sciences, Eli Lilly to 40%) [1–4] . New advances in mAb down- recent years, flocculation-based pretreatment & Company, 450 East 29th Street, stream processing are critically needed, in of cell cultures to circumvent both harvest New York, NY 10016, USA order to meet the significant challenges posed and purification issues experienced in mAb by increased cell culture productivity.
    [Show full text]
  • Downstream Processing
    Chapter 11 Downstream Processing ©2016 Montgomery County Community College Objectives This chapter provides an overview of downstream processing. After completing this chapter, students will be able to: . Describe the general elements of the downstream purification process for a typical monoclonal antibody-based therapeutic, starting with the bioreactor harvest pool and finishing with pre-formulation, pure drug product. Explain the overall goals of the purification process from the standpoint of purity, yield, and efficiency as well as the general strategies employed to achieve these goals. Define the specific methods and in-process tools and materials used in typical steps of the downstream process and the purposes and physical/chemical mechanisms occurring at each step, including: – clarification of the bioreactor harvest by depth filtration and/or centrifugation – capture chromatography for product concentration and initial purification – ultrafiltration and diafiltration of the product at various stages in the downstream process – intermediate purification chromatography steps – final polishing chromatography steps . Describe general economic factors contributing to the cost of goods produced and common considerations/strategies for optimizing and improving those process economics. Classify specific departmental contributions to the ongoing operation and maintenance of the downstream unit operations within a facility. 424 Chapter 11 - Downstream Processing Terms Capture/recovery: the rapid separation of the product of interest from
    [Show full text]
  • Development of Aqueous Two-Phase Separations by Combining High-Throughput Screening and Process Modelling
    Development of aqueous two-phase separations by combining high-throughput screening and process modelling A thesis submitted to University College London for the degree of DOCTOR OF ENGINEERING by Nehal Patel Friday 21st July 2017 The Advanced Centre for Biochemical Engineering Department of Biochemical Engineering University College London Gower Street, London, WC1E 6BT, UK 1 I, Nehal Patel, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract Separation based on aqueous two-phase extraction (ATPE) is a promising downstream separation technology for the production of biological products. The advantages of using aqueous two-phase systems include but are not limited to easy scalability, ease of continuous operation and a favourable environment for biological compounds. One of the main challenges associated with aqueous two-phase systems is process development. This is in part due to the many factors which influence the separation of biological materials in such systems such as polymer and salt type, pH and charge. The large number of factors to consider makes the development of aqueous two-phase systems challenging due to the need to find a robust and efficient separation in a large experimental space. This work addresses this issue by considering the use of dynamic process models and high-throughput experimentation for the development of aqueous two-phase extraction processes for biological products. The use of a dynamic equilibrium stage process model to simulate aqueous two-phase extraction is considered in Chapter 3. The process model is capable of simulating various modes of operation; and both multi-cycle batch and continuous counter-current modes of operation are considered.
    [Show full text]
  • Desalting and Buffer Exchange by Dialysis, Gel Filtration Or Diafiltration*
    Scientific & Technical Report PN 33290 Desalting and Buffer Exchange by Dialysis, Gel Filtration or Diafiltration* By Larry Schwartz, Senior Technical Manager, Pall Life Sciences DIALYSIS GEL FILTRATION Dialysis is an old established procedure for Gel Filtration is a non-adsorptive chromatography reducing the salt concentration in samples. technique that separates molecules on the basis It requires filling a dialysis bag (membrane of molecular size. Desalting and buffer exchange casing of defined porosity), tying the bag off, are two special examples of gel filtration that and placing the bag in a bath of water or are widely used in many downstream bio- buffer. Through diffusion, the concentration of processes. Desalting is used to completely salt in the bag will equilibrate, with that in the remove or lower the concentration of salt or bath. Large molecules that can’t diffuse other low molecular weight components in the through the bag remain in the bag. If the bath sample while buffer exchange replaces the is water, the concentration of the small mole- sample buffer with a new buffer. cules in the bag will decrease slowly until the Gel filtration is one of the easiest chromatography concentration inside and outside are the same. methods to perform because samples are The greater the volume of the bath relative to processed using isocratic elution. In its the sample volume in the bags, the lower the analytical form, gel filtration (also known as equilibration concentration that can be reached. size exclusion chromatography) can distinguish Usually several replacements of the bath water between molecules (e.g. proteins) with a are required to completely remove all of the molecular weight difference of less than a salt.
    [Show full text]
  • Diffusion Using Dialysis Tubing
    Diffusion Using Dialysis Tubing Introduction SCIENTIFIC How do membranes around cells regulate the internal composition of the cell? Can any molecule BIO FAX! pass through the membrane or only certain molecules. What determines the direction of molecular movement? Use dialysis tubing to teach students the fundamental concepts of diffusion. Concepts •Diffusion • Concentration gradient • Semipermeable membrane Background Diffusion is one of the key processes involved in the movement of materials throughout living systems. Diffusion is the tendency for molecules of any substance to spread out into the available space. Diffusion can be effectively demonstrated by observing the action of a drop of dye in a glass of water. The H2O molecules that make up water are in constant motion. If a drop of blue food dye is added to the glass, the dye begins to slowly diffuse throughout the water. The individual dye molecules disperse and collide with the moving water molecules and eventually become evenly distributed throughout the glass. The solution in the glass appears as a uniform light blue. The movement of individual molecules is indeed random. The net movement of the dye molecules is directional in a sense that they move from a region where they are highly concentrated to a region where they are less concentrated. Diffusion is said to occur down a concentration gradient. When the dye has become evenly distributed, and the dye and water are at equilibrium, the net movement of the dye appears to have stopped. It is important to understand that the random movement of individual dye and water molecules continues even when equilibrium is reached.
    [Show full text]