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Hydrocyclones for the separation of yeast and protein particlesYuan, Huixin January 1996 (has links)
No description available.
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Novel Methods to Construct Microchannel Networks with Complex TopologiesHuang, Jen-Huang 14 March 2013 (has links)
Microfluidic technology is a useful tool to help answer unsolved problems in multidisciplinary fields, including molecular biology, clinical pathology and the pharmaceutical industry.Current microfluidic based devices with diverse structures have been constructed via extensively used soft lithography orphotolithography fabrication methods. A layer-by-layer stacking of 2D planar microchannel arrays can achieve limited degrees of three dimensionality. However, assembly of large-scale multi-tiered structures is tedious, and the inherently planar nature of the individual layers restricts the network’s topological complexity. In order to overcome the limitations of existing microfabrication methodswe demonstrate several novel methods that enable microvasculature networks: electrostatic discharge,global channel deformation and enzymatic sculpting to fabricate complex surface topologies.
These methods enable construction of networks of branched microchannels arranged in a tree-like architecture with diameters ranging from approximately 10 μm to 1 mm. Interconnected networks with multiple fluidic access points can be straightforwardly constructed, and quantification of their branching characteristics reveals remarkable similarity to naturally occurring vasculature. In addition, by harnessing enzymatic micromachining we are able to construct nanochannels, microchannels containing embedded features templated by the substrate’s crystalline morphology, and an irregular cross section of microchannel capable of performing isolation and enrichment of cells from whole blood with throughput 1 – 2 orders of magnitude faster than currently possible. These techniques can play a key role in developing an organ-sized engineered tissue scaffolds and high-throughput continuous flow separations.
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HYDROGEL BASED MEMBRANES FOR BIOPHARMACEUTICAL AND BIOMEDICAL APPLICATIONSYOO, SEUNG MI January 2014 (has links)
Membrane technology has been actively used as a separation tool in the chemical, environmental, and biopharmaceutical industries for several decades. As membrane quality requirement in the industry has increased, efforts have been directed towards enhancement in mechanical strength, chemical durability and functionality of membranes. One of the approaches for membrane quality enhancement is based on the combination of hydrogel technology with membrane technology. This thesis focused on the application and development of hydrogel based membranes, notably hydrophilized PVDF (polyvinylidene fluoride) membrane for hydrophobic interaction membrane chromatography; the fabrication of paper-hydrogel composite membranes for membrane chromatography; development of a technique for coating alginate (a natural hydrogel) on the outer surface of a hollow fiber membrane for potential application in bioreactors and the use of hollow fiber membranes as mold for fabrication calcium alginate fibers for biomedical and tissue engineering applications.
A membrane chromatography-based polishing technique was developed for removing leached protein-A and aggregates from monoclonal antibody (mAb). A commercial synthetic membrane that is known to be hydrophilized by hydrogel grafting was employed to develop this polishing process that resulted in highly pure mAb, free from aggregates and protein-A. This mAb polishing technique could easily be integrated with a hydrophobic interaction membrane chromatography based mAb purification process.
A paper-hydrogel composite membrane was developed as an inexpensive alternative to commercial synthetic membranes used for carrying hydrophobic interaction membrane chromatography. Poly(N-vinylcaprolactam) or PVCL hydrogel was coated on Whatman filter paper to prepare these membranes. These environment responsive membrane which responded to changes in salt concentration, gave excellent fractionation of multi-component protein mixtures. As case study, a mixture of immunoglobulin G, human serum albumin and insulin was fractionated.
A technique for modifying the surface of synthetic hollow fiber membranes with alginate (a natural hydrogel) was developed. This manner of surface modification led to the improvement in membrane mass transport. The alginate was cross-linked on the outer surface of the membrane by diffusion of the cross-linker (calcium ions) through the membrane pores. The calcium alginate coating layer was characterized by optical and transmission electron microscopy, contact angle measurement, hydraulic permeability measurement and by examining solute transport.
Hollow and solid calcium alginate fibers were fabricated using a novel hollow fiber membrane based moulding technique. The pore present on the hollow fiber membrane served as the reservoir for the calcium chloride solution with cross-linked the alginate within the lumen. The calcium alginate fibers produced were characterized by optical, transmission electron, and scanning electron microscopy. Cell immobilization experiments were carried out to demonstrate biocompatibility and potential for tissue engineering applications. / Thesis / Doctor of Philosophy (PhD)
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Synthesis and Characterization of Environment-Responsive Membranes for Bioseparations / Environment-Responsive Membranes for BioseparationsHuang, Ruixiang 08 1900 (has links)
Environment-responsive membranes were created by modification of a commerical polyvinylidene fluoride (PVDF) membrane support with a thermo-responsive hydrogel composed of poly N-vinyllactams cross-linked with bisacrylamide. The modified membranes were then characterized by their percentage mass gains as well as by their valve effect in response to changes in salt concentration. One set of membranes, with a large valve effect, was selected for highest retention of intermediately sized proteins was examined for ultrafiltration-based protein separation applications. A batch separation protocol featuring pulsed sample injection technique (PSIT) was then used to sieve single proteins and to fractionate a synthetic binary protein mixture and a synthetic ternary protein mixture with some success, demonstrating the potential of these environment-responsive membranes for use in multi-component separations. A second set of membranes, with a small valve effect, was selected for its ability to alter between hydrophobic and hydrophilic states under different environmental conditions and its potential in hydrophobic interaction membrane chromatography (HIMC) applications was successfully demonstrated by comparing against a benchmark membrane that is used successfully for HIMC applications in prior literature. / Thesis / Master of Applied Science (MASc)
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Protein Bioseparation using Synthetic Membranes: Enhancement of Selectivity and ThroughputKanani, Dharmeshkumar M. January 2007 (has links)
Cost-effective large-scale protein bioseparation will be the key issue for the biopharmaceutical industry in the coming years. Conventional protein purification techniques are severely limited in the sense that they give either good selectivity of separation at the cost of throughput or vice versa. Synthetic membrane based bioseparation techniques such as high-resolution ultrafiltration and membrane chromatography have the potential to combine high-throughput with high selectivity. This thesis focuses on approaches for obtaining both selectivity and throughput in membrane based protein bioseparation processes.
Obtaining high selectivity is one of the main objectives in high-resolution ultrafiltration. This thesis reports a novel approach for flexibly manipulating the selectivity of protein separation using a dual-facilitating agent. In this study it has been shown for the first time that the selectivity of separation can be altered as desired, i.e. if required, the selectivity can be reversed and thereby smaller proteins can be retained and larger proteins can be made to permeate by using a dual-facilitating agent. The results are explained in terms of protein-protein electrostatic interactions and Donnan effect. This novel approach is expected to significantly increase the flexibility of carrying out high-resolution ultrafiltration.
Membrane chromatography is based on the use of stacks of microporous synthetic membranes as chromatographic media. Due to lower binding capacities of commercial membranes in comparison to conventional beads for packed bed chromatography, the
commercial success of membrane chromatography is largely limited to the flow-through applications. The study on membrane chromatography addresses the performances of new types of high-capacity macroporous gel-filled membranes for ion-exchange chromatography of proteins. This work demonstrates the suitability of using one of these novel membranes for fractionation of plasma proteins.
Membrane fouling reduces product throughput and is considered a major problem in pressure driven membrane processes such as microfiltration and ultrafiltration. This thesis reports some significant contributions in the area of membrane fouling. A novel yet conceptually simple approach for modeling flux decline in constant pressure ultrafiltration, which takes into account the interplay between flux, concentration polarization and membrane fouling is discussed. Conventional fouling models account for the effects of concentration polarization and membrane fouling in a simple additive way. The basic hypothesis in the model discussed here is that flux decline in constant pressure ultrafiltration is self-attenuating in nature. This new approach is expected to be very useful in deciding the start-up conditions in membrane processes. Despite widespread use of in-line microfiltration for sterilization of therapeutic proteins prior to formulation, there has been no systematic study on fouling in such processes. Part of the fouling work in this thesis examines how resistance to filtration increases during in-line microfiltration of concentrated protein solution and the mechanism of protein fouling. It assesses the severity of fouling in terms of apparent reversible fouling and irreversible fouling. Traditional methods to measure the protein fouling resistances of membranes are time consuming and expensive. This thesis reports three protocols to compare the performance of microfiltration membranes for protein filtration. The first protocol, which is based on accelerated fouling in the dead end mode using pulsed injection technique is rapid, simple, and cost effective and gives valuable information about membrane performance. The remaining two protocols are based on the critical flux concept. / Thesis / Doctor of Philosophy (PhD)
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Investigation of the Interactions between Biomolecules and Mesoporous Inorganic Materials in Biomolecule Immobilization for Bioseparation and BiocatalysisKim, Jungseung January 2011 (has links)
No description available.
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Functionalized corundum as a novel affinity platform for the efficient purification of proteins from complex biological samplesVölzke, Jule Lexa 10 January 2024 (has links)
Diese Arbeit hatte es zum Ziel, eine neue und effiziente Affinitätsplattform für die Aufreinigung und Isolierung von Proteinen basierend auf nicht‐porösen und stabilen Korund‐Partikeln zu entwickeln. Das Rohmaterial wurde kovalent mit Proteinbindern modifiziert, um so die Isolierung spezieller Target‐Proteine aus komplexen biologischen Proben zu realisieren. Für die erste Modifizierungsschicht auf der Korundoberfläche wurden verschiedene Phosphonsäuren und Silane untersucht. Anschließend wurde der etablierte bifunktionale Crosslinker Glutaraldehyd mit dem biokompatibleren verzweigten Polyglycerol (PG) verglichen. Es konnte gezeigt werden, dass Polyglycerol eine hervorragende Alternative zu Glutaraldehyd darstellen kann. Für die angestrebte Anwendung als neues Tool für die Antikörperreinigung wurde humanes IgG mit Protein‐A‐funktionalisierten Korundpartikeln erfolgreich aus Humanplasma isoliert. Um das Anwendungsspektrum der neuen Korundmethode auszuweiten, wurde im zweiten Teil dieser Arbeit eine Affinitätsplattform für die Isolierung Polyhistidin‐getaggten Proteinen basierend auf Metallionen‐funktionalisiertem Korund entwickelt und angewendet. Hauptkriterien dieser Methodenentwicklung waren die schnelle, effiziente und ökonomische Aufreinigung rekombinanter Proteine aus bakteriellen Lysaten in einem säulenfreien Format. Als Modellsystem wurde Polyhistidin‐getaggtes Protein A/G (His6‐PAG) mit Rinderserumalbumin versetzt, was die Entwicklung eines optimierten Protokolls für die Isolierung rekombinanter Proteine ermöglichte. Im direkten Vergleich mit kommerziellen Ni‐NTA‐Agarose‐Beads zeigten die Korundpartikel höhere Proteinreinheiten. Abschließend konnte gezeigt werden, dass Zink eine ideale Alternative als Metallion darstellt, um etwaige Nachteile des Nickels in der Anwendung von Life‐Science‐Methoden zu umgehen und eine zukunftsträchtige Methode mit weiterem Potenzial zu realisieren. / This work aimed to develop and establish a novel efficient affinity platform for protein purification based on nonporous, stable, and easily available corundum powder. The material was functionalized covalently with protein binders to isolate and enrich specific proteins from complex biological matrices. Phosphonic acids and silanes were tested as the first modification layer. In the next step, the well‐known bifunctional crosslinker glutaraldehyde was compared with a more biocompatible, hyperbranched polyglycerol (PG). It could have been shown that oxidized polyglycerol is an excellent alternative to glutaraldehyde. Human IgG was purified with protein A functionalized corundum from crude human plasma for the initial purpose of antibody isolation. To broaden the application of functionalized corundum, a novel purification platform for the isolation of poly His‐tagged proteins based on metal‐ion‐functionalized corundum was established.The main goal of this approach was the efficient, economical, and fast purification of recombinant proteins in a column‐free format that can also easily be performed in moderately equipped laboratories. As a model system for method optimization, His‐tagged protein A/G (His6‐PAG), mixed with bovine serum albumin (BSA), was established leading to a purification protocol with minimal nonspecific binding by the variation of the imidazole content in the used binding and washing buffers. Corundum in direct comparison with standard Ni‐NTA agarose beads generated higher purities of isolated proteins. Finally, it was shown that zinc‐functionalized corundum is another efficient approach to circumvent potential negative effects of nickel use in life science applications.
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Study of the Preparation of Mesoporous Magnetic Microspheres and Their ApplicationsEricson, Mårten January 2009 (has links)
Treatment of wastewater using magnetic technology is a rising field. In this thesis, the latest research on the subject is reviewed and several adsorbents with different coatings, which impart them unique properties, are discussed. Separation of particles from aqueous solution using magnetic technology is more convenient compared to conventional techniques, such as filtration and centrifugation. The adsorbents described in this thesis are effective for adsorption of several types of contaminants, such as heavy metals and different types of dyes. Magnetic microspheres were synthesised using porous polystyrene microspheres as template. The microspheres were first sulfonated using chlorosulfonic acid followed by stirring in the presence of ferrous chloride which then was oxidised and magnetic nanoparticles were formed on the surface. The sulfonated microspheres had a surface area of 420 m2/g and the magnetic 175 m2/g, indicative of Fe3O4 nanoparticles were successfully formed in the pores. The weight fraction of the Fe3O4 nanoparticles in the magnetic microspheres was 33 %. Adsorption and desorption studies of the cationic dye, methylene blue, using mesoporous magnetic microspheres were performed. The results show that the mesoporous magnetic microspheres have good ability to adsorb methylene blue at low concentrations. In a cycle study the adsorption efficiency were nearly 100 % throughout the study. Using a 6/4 EtOH/H2O with saturated KCl solution the desorption efficiency in the cycle study were about 95 %. The microspheres were used as carriers for TiO2 in order to overcome the problem with the separation of TiO2 from solution. The TGA results show that the microspheres contained about 12 % of TiO2. The TiO2 coated microspheres were used for the photocatalytic degradation of phenol. However, the TiO2 microspheres did not work. This was a result from that the phenol had too little contact with the TiO2. A possible way of solving this problem could be to decrease the size of the microspheres, thus increase the surface area. Lysozyme was adsorbed and separated using the porous microspheres. The lysozyme adsorption worked best at pH 9.6, which is the pI for lysozyme. The lysozyme could be extracted from the microspheres by using a pH 13 buffer. Also, by using MeOH/H2O and EtOH/H2O solutions with saturated KCl the lysozyme could be desorbed. An adsorption and desorption mechanism was also presented. / Vattenrening med magnetisk teknologi är en ny och alltmer uppmärksammad teknik. Magnetisk separation är ett enkelt och snabbt sätt att separera något från en lösning. Magnetisk separation är mer lätthanterligt jämfört med traditionell separationsteknik såsom centrifugering och filtrering. Med porösa polystyren mikrosfärer som mall, syntetiserades magnetiska mikrosfärer. Först så sulfonerades mikrosfärerna med klorosulfonisk syra, följt av att de rördes om i en järnkloridlösning. Magnetiska nanopartiklar bildades i porerna och på ytan av mikrosfärerna. Sulfonerade mikrosfärerna hade en specifik ytarea på 420 m2/g och de magnetiska 175 m2/g, detta indikerar att Fe3O4-nanopartiklar bildades på ytan och i porerna. Massfraktionen av Fe3O4 var 33 %. Adsorption- och desorptionsstudier på de magnetiska mikrosfärerna utfördes. Färgämnet metylblått användes i studien. Resultaten visade att magnetiska mikrosfärerna hade en bra adsorptionsförmåga vid låga koncentrationer av metylblått. Cykelstudier visade att adsorptionsverkningsgraden var nära 100 % under flera adsorptionscykler. Desorptionsförsök med olika lösningsmedel visade att en mättad KCl 6/4 EtOH/H2O lösning gav en desorptions-verkningsgrad på ca 95 %. Mikrosfärerna användes som mall och kärna för att syntetisera en TiO2-fotokatalysator, detta för att överkomma problemet som finns med separation av rent TiO2 pulver från lösning. TGA resultaten visade att mikrosfärerna innehöll ca 12 % TiO2. De syntetiserade TiO2-mikrosfärerna användes till att bryta ner fenol fotokatalytiskt. Dock fungerade inte detta experiment. En anledning var att fenolen hade för lite kontakt med TiO2. En lösning på detta problem är att använda mikrosfärer med högre specifik ytarea. Proteinet lysozym användes som modellprotein för försök att separera proteiner från lösning genom att använda porösa mikrosfärer. Resultatet visade att lysozym kunde adsorberas vid pH 9.6. Med en pH 13 buffer kunde lysozymet sedan extraheras från mikrosfärerna. En mekanism för adsorptionen och desorptionen på mikrosfärerna presenterades.
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Fractionation of non-animal protein hydrolysates for use in Chinese Hamster Ovary cell mediaYoo, Seung Mi 22 January 2010 (has links)
This thesis presents a study on the enhancement of CHO cell growth by Yeast extract, Yeastolate, and Primatone fractions obtained by dead-end ultrafiltration. The total solid, peptide contents, antioxidant capacity and hydrophobicity of the fractions were evaluated.
The objective of this project was to evaluate the potential of sequential ultrafiltration as an effective, simple and economical method for the identification of CHO cell growth enhancement components in yeast extract and yeastolate (primatone).
The fractionation by sequential ultrafiltration (50 kDa membrane, 3 kDa membrane and 1 kDa membrane) of yeast extract (YE), yeastolate (YET), and primatone (PRI) showed different fouling and fractionation behaviour. Significant fouling was observed with the 50 kDa and 3 kDa membrane while negligible fouling was observed with the 1 kDa membrane. Similar and more significant fouling was observed with the 50 kDa membrane and for YE and PRI in comparison to YET. In contrast, more fouling was observed during the ultrafiltration with the 3kDa MWCO and for YE and YET in comparison to PRI. Finally a relatively constant permeate flux was obtained with the 1 kDa membrane, with PRI the highest and YET the lowest permeate flux. Different total peptide contents were present in the three feeds, 410, 327 and 300 mmol Phe-Gly equivalent/ g total solids for YE, PRI and YET respectively. In spite of different feed equivalent Phe-Gly, all three feeds contained a similar amount of equivalent Phe-Gly with molecular weight larger than 50 kDa, 15-19% of the initial feed stream. This was similar amount to the total solids content. The total peptide content of the retentate obtained for the 3kDa filtration indicated that YE and YET contained ~ 20% of equivalent Phe-Gly larger than 3 kDa but smaller than 50kDa. In contrast, PRI contained only 6% of equivalent Phe-Gly with such molecular weight. The retentate of the 1kDa filtration contained 55% of the feed equivalent Phe-Gly compared to 47% for YE and 38% for YET (p< 0.05). All three feeds have similar total peptide content smaller than 1 kDa. For any given feed, the equivalent Phe-Gly was larger than 1 kDa but smaller than 3 kDa predominated. The total peptide content profile according to size coincides with the total solids distribution for all three feed types. This is the first study that reports on the total peptide content for YE, YET, and PRI subjected to ultrafiltration fractionation.
All three feeds and their fractions when freeze-dried had similar antioxidant capacity estimated by the FCR (Folin-Ciocalteu reagent) assay, ~ 40-50 mg Trolox/g sample.
The bioactivity of feed and fractions was measured as cell density for CHO (beta-IFN producers) in basal medium supplemented with a combination of the crude non-fractionated feed material and a specific fraction and grown in T25 flasks. PRI showed a similar growth enhancement effect for all fractions when compared to a culture supplemented with the crude non-fractionated. YE showed no growth enhancement for any of the fractions when compared to a culture supplemented with the crude non-fractionated YE. This observation need to be confirmed as a culture supplemented with the crude non-fractionated YE showed a very high growth stimulating effect which was much higher than PRI and YET at the same concentration. Finally, YET 3kDa retentate fraction displayed a 50 % growth enhancement effect. In conclusion, the fractions obtained from the two non-animal protein hydrolysates considered in this study, YE and YET showed limited CHO cell growth enhancement effect when compared to the non-fractionated material. Only the YET 3kDa retentate fraction displayed a good CHO cell growth enhancement effect. YET 3kDa represent an attractive serum substitute for its use in culturing CHO cells. PRI, an animal derived protein hydrolysate showed the best growth enhancement effect for all fractions produced in this study. These results suggest that YET has high potential as a media additive for the development of serum-free media which can promote cell growth and, in the future this work can contribute in production of therapeutic proteins markets.
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Fractionation of non-animal protein hydrolysates for use in Chinese Hamster Ovary cell mediaYoo, Seung Mi 22 January 2010 (has links)
This thesis presents a study on the enhancement of CHO cell growth by Yeast extract, Yeastolate, and Primatone fractions obtained by dead-end ultrafiltration. The total solid, peptide contents, antioxidant capacity and hydrophobicity of the fractions were evaluated.
The objective of this project was to evaluate the potential of sequential ultrafiltration as an effective, simple and economical method for the identification of CHO cell growth enhancement components in yeast extract and yeastolate (primatone).
The fractionation by sequential ultrafiltration (50 kDa membrane, 3 kDa membrane and 1 kDa membrane) of yeast extract (YE), yeastolate (YET), and primatone (PRI) showed different fouling and fractionation behaviour. Significant fouling was observed with the 50 kDa and 3 kDa membrane while negligible fouling was observed with the 1 kDa membrane. Similar and more significant fouling was observed with the 50 kDa membrane and for YE and PRI in comparison to YET. In contrast, more fouling was observed during the ultrafiltration with the 3kDa MWCO and for YE and YET in comparison to PRI. Finally a relatively constant permeate flux was obtained with the 1 kDa membrane, with PRI the highest and YET the lowest permeate flux. Different total peptide contents were present in the three feeds, 410, 327 and 300 mmol Phe-Gly equivalent/ g total solids for YE, PRI and YET respectively. In spite of different feed equivalent Phe-Gly, all three feeds contained a similar amount of equivalent Phe-Gly with molecular weight larger than 50 kDa, 15-19% of the initial feed stream. This was similar amount to the total solids content. The total peptide content of the retentate obtained for the 3kDa filtration indicated that YE and YET contained ~ 20% of equivalent Phe-Gly larger than 3 kDa but smaller than 50kDa. In contrast, PRI contained only 6% of equivalent Phe-Gly with such molecular weight. The retentate of the 1kDa filtration contained 55% of the feed equivalent Phe-Gly compared to 47% for YE and 38% for YET (p< 0.05). All three feeds have similar total peptide content smaller than 1 kDa. For any given feed, the equivalent Phe-Gly was larger than 1 kDa but smaller than 3 kDa predominated. The total peptide content profile according to size coincides with the total solids distribution for all three feed types. This is the first study that reports on the total peptide content for YE, YET, and PRI subjected to ultrafiltration fractionation.
All three feeds and their fractions when freeze-dried had similar antioxidant capacity estimated by the FCR (Folin-Ciocalteu reagent) assay, ~ 40-50 mg Trolox/g sample.
The bioactivity of feed and fractions was measured as cell density for CHO (beta-IFN producers) in basal medium supplemented with a combination of the crude non-fractionated feed material and a specific fraction and grown in T25 flasks. PRI showed a similar growth enhancement effect for all fractions when compared to a culture supplemented with the crude non-fractionated. YE showed no growth enhancement for any of the fractions when compared to a culture supplemented with the crude non-fractionated YE. This observation need to be confirmed as a culture supplemented with the crude non-fractionated YE showed a very high growth stimulating effect which was much higher than PRI and YET at the same concentration. Finally, YET 3kDa retentate fraction displayed a 50 % growth enhancement effect. In conclusion, the fractions obtained from the two non-animal protein hydrolysates considered in this study, YE and YET showed limited CHO cell growth enhancement effect when compared to the non-fractionated material. Only the YET 3kDa retentate fraction displayed a good CHO cell growth enhancement effect. YET 3kDa represent an attractive serum substitute for its use in culturing CHO cells. PRI, an animal derived protein hydrolysate showed the best growth enhancement effect for all fractions produced in this study. These results suggest that YET has high potential as a media additive for the development of serum-free media which can promote cell growth and, in the future this work can contribute in production of therapeutic proteins markets.
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