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Released this past November, the list is based on data collected from the Web of Science and highlights some of the world’s most influential scientific minds by naming the researchers whose publications over the previous decade have included a high number of Highly Cited Papers placing them among the top 1% most-cited.
\\n\\nWe wish to congratulate all of the researchers named and especially our authors on this amazing accomplishment! We are happy and proud to share in their success!
\\n"}]',published:!0,mainMedia:null},components:[{type:"htmlEditorComponent",content:'IntechOpen is proud to announce that 179 of our authors have made the Clarivate™ Highly Cited Researchers List for 2020, ranking them among the top 1% most-cited.
\n\nThroughout the years, the list has named a total of 252 IntechOpen authors as Highly Cited. Of those researchers, 69 have been featured on the list multiple times.
\n\n\n\nReleased this past November, the list is based on data collected from the Web of Science and highlights some of the world’s most influential scientific minds by naming the researchers whose publications over the previous decade have included a high number of Highly Cited Papers placing them among the top 1% most-cited.
\n\nWe wish to congratulate all of the researchers named and especially our authors on this amazing accomplishment! We are happy and proud to share in their success!
\n'}],latestNews:[{slug:"stanford-university-identifies-top-2-scientists-over-1-000-are-intechopen-authors-and-editors-20210122",title:"Stanford University Identifies Top 2% Scientists, Over 1,000 are IntechOpen Authors and Editors"},{slug:"intechopen-authors-included-in-the-highly-cited-researchers-list-for-2020-20210121",title:"IntechOpen Authors Included in the Highly Cited Researchers List for 2020"},{slug:"intechopen-maintains-position-as-the-world-s-largest-oa-book-publisher-20201218",title:"IntechOpen Maintains Position as the World’s Largest OA Book Publisher"},{slug:"all-intechopen-books-available-on-perlego-20201215",title:"All IntechOpen Books Available on Perlego"},{slug:"oiv-awards-recognizes-intechopen-s-editors-20201127",title:"OIV Awards Recognizes IntechOpen's Editors"},{slug:"intechopen-joins-crossref-s-initiative-for-open-abstracts-i4oa-to-boost-the-discovery-of-research-20201005",title:"IntechOpen joins Crossref's Initiative for Open Abstracts (I4OA) to Boost the Discovery of Research"},{slug:"intechopen-hits-milestone-5-000-open-access-books-published-20200908",title:"IntechOpen hits milestone: 5,000 Open Access books published!"},{slug:"intechopen-books-hosted-on-the-mathworks-book-program-20200819",title:"IntechOpen Books Hosted on the MathWorks Book Program"}]},book:{item:{type:"book",id:"9405",leadTitle:null,fullTitle:"Quinazolinone and Quinazoline Derivatives",title:"Quinazolinone and Quinazoline Derivatives",subtitle:null,reviewType:"peer-reviewed",abstract:"One of the problems with modern public health is target searching for new highly effective medicinal preparations. Among those medicinal preparations are the natural and synthetic origins of quinazolinone-4 derivatives. Quinazolinone derivatives are reported to be physiologically and pharmacologically active. They also exhibit a wide range of activities such as anticonvulsant, antiinflammatory, antifungal, antimalarial, and sedative properties. Some of these compounds are identified as drugs used as diuretics, vasodilators, and antihypertensive agents. Moreover, sulfonamide derivatives have been widely used as bacteriostatic agents. Prompted by the above-mentioned facts and in conjunction with our ongoing program on the utility of readily obtainable starting material for the synthesis of heterocyclic systems of biological interest, we have decided to synthesize a series of quinazolinone derivatives having sulfonamide moiety with a potentially wide spectrum of biological responses.",isbn:"978-1-83880-140-3",printIsbn:"978-1-83880-139-7",pdfIsbn:"978-1-83880-053-6",doi:"10.5772/intechopen.85315",price:119,priceEur:129,priceUsd:155,slug:"quinazolinone-and-quinazoline-derivatives",numberOfPages:126,isOpenForSubmission:!1,isInWos:null,hash:"95a736fcc80804d0875730b3515aa659",bookSignature:"Ali Gamal Al-kaf",publishedDate:"May 6th 2020",coverURL:"https://cdn.intechopen.com/books/images_new/9405.jpg",numberOfDownloads:1853,numberOfWosCitations:0,numberOfCrossrefCitations:1,numberOfDimensionsCitations:2,hasAltmetrics:0,numberOfTotalCitations:3,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"March 25th 2019",dateEndSecondStepPublish:"September 23rd 2019",dateEndThirdStepPublish:"November 22nd 2019",dateEndFourthStepPublish:"February 10th 2020",dateEndFifthStepPublish:"April 10th 2020",currentStepOfPublishingProcess:5,indexedIn:"1,2,3,4,5,6,7",editedByType:"Edited by",kuFlag:!1,editors:[{id:"191580",title:null,name:"Ali Gamal",middleName:null,surname:"Al-kaf",slug:"ali-gamal-al-kaf",fullName:"Ali Gamal Al-kaf",profilePictureURL:"https://mts.intechopen.com/storage/users/191580/images/system/191580.jpeg",biography:"PhD. in pharmaceutical sciences from Russia 2006, Chief Council for Accreditation and Quality Assurance. Previous Dean of Faculty of Pharmacy at Sana\\'a University. Professor of Medicinal Chemistry Department. Member of Yemeni Medical Council. Member of many associations and international groups. Executive Editor in Universal Journal of Pharmaceutical Research. Editor and Associate Editor of some international journals. My interest is synthesis and biological activity of 4-oxopyrimidine and quinazolinone -4 derivatives, Structural biology and bioinformatics in drug design, study of Yemeni medicinal plants , development and validation of Spectrophotometric and HPLC methods for different drugs and antibiotics and antimicrobial resistance in Yemen.\nAuthor of more than 70 publications, 4 patents and 11 books.",institutionString:"Sana'a University",position:null,outsideEditionCount:0,totalCites:0,totalAuthoredChapters:"3",totalChapterViews:"0",totalEditedBooks:"3",institution:{name:"Sana'a University",institutionURL:null,country:{name:"Yemen"}}}],equalEditorOne:null,equalEditorTwo:null,equalEditorThree:null,coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"85",title:"Organic Chemistry",slug:"organic-chemistry"}],chapters:[{id:"68832",title:"Introductory Chapter: The Newest Research in Quinazolinone and Quinazoline Derivatives",doi:"10.5772/intechopen.88913",slug:"introductory-chapter-the-newest-research-in-quinazolinone-and-quinazoline-derivatives",totalDownloads:183,totalCrossrefCites:0,totalDimensionsCites:0,signatures:"Ali Gamal Al-kaf",downloadPdfUrl:"/chapter/pdf-download/68832",previewPdfUrl:"/chapter/pdf-preview/68832",authors:[{id:"191580",title:null,name:"Ali Gamal",surname:"Al-kaf",slug:"ali-gamal-al-kaf",fullName:"Ali Gamal Al-kaf"}],corrections:null},{id:"70910",title:"Biological Activity of Quinazolinones",doi:"10.5772/intechopen.90621",slug:"biological-activity-of-quinazolinones",totalDownloads:345,totalCrossrefCites:0,totalDimensionsCites:0,signatures:"Awwad A. 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The contents of the book will be written by multiple authors and edited by experts in the field.",isbn:null,printIsbn:null,pdfIsbn:null,doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!1,hash:"e4f2d361bf0521f27d50aab719db5045",bookSignature:"",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/9858.jpg",keywords:null,numberOfDownloads:null,numberOfWosCitations:0,numberOfCrossrefCitations:null,numberOfDimensionsCitations:null,numberOfTotalCitations:null,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"June 6th 2019",dateEndSecondStepPublish:"June 27th 2019",dateEndThirdStepPublish:"August 26th 2019",dateEndFourthStepPublish:"November 14th 2019",dateEndFifthStepPublish:"January 13th 2020",remainingDaysToSecondStep:"2 years",secondStepPassed:!0,currentStepOfPublishingProcess:1,editedByType:null,kuFlag:!1,biosketch:null,coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:null,coeditorOne:null,coeditorTwo:null,coeditorThree:null,coeditorFour:null,coeditorFive:null,topics:[{id:"10",title:"Earth and Planetary Sciences",slug:"earth-and-planetary-sciences"}],chapters:null,productType:{id:"1",title:"Edited Volume",chapterContentType:"chapter",authoredCaption:"Edited by"},personalPublishingAssistant:null},relatedBooks:[{type:"book",id:"5962",title:"Estuary",subtitle:null,isOpenForSubmission:!1,hash:"43058846a64b270e9167d478e966161a",slug:"estuary",bookSignature:"William Froneman",coverURL:"https://cdn.intechopen.com/books/images_new/5962.jpg",editedByType:"Edited by",editors:[{id:"109336",title:"Prof.",name:"William",surname:"Froneman",slug:"william-froneman",fullName:"William Froneman"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"1591",title:"Infrared Spectroscopy",subtitle:"Materials Science, Engineering and Technology",isOpenForSubmission:!1,hash:"99b4b7b71a8caeb693ed762b40b017f4",slug:"infrared-spectroscopy-materials-science-engineering-and-technology",bookSignature:"Theophile Theophanides",coverURL:"https://cdn.intechopen.com/books/images_new/1591.jpg",editedByType:"Edited by",editors:[{id:"37194",title:"Dr.",name:"Theophanides",surname:"Theophile",slug:"theophanides-theophile",fullName:"Theophanides Theophile"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3161",title:"Frontiers in Guided Wave Optics and Optoelectronics",subtitle:null,isOpenForSubmission:!1,hash:"deb44e9c99f82bbce1083abea743146c",slug:"frontiers-in-guided-wave-optics-and-optoelectronics",bookSignature:"Bishnu Pal",coverURL:"https://cdn.intechopen.com/books/images_new/3161.jpg",editedByType:"Edited by",editors:[{id:"4782",title:"Prof.",name:"Bishnu",surname:"Pal",slug:"bishnu-pal",fullName:"Bishnu Pal"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"3092",title:"Anopheles mosquitoes",subtitle:"New insights into malaria vectors",isOpenForSubmission:!1,hash:"c9e622485316d5e296288bf24d2b0d64",slug:"anopheles-mosquitoes-new-insights-into-malaria-vectors",bookSignature:"Sylvie Manguin",coverURL:"https://cdn.intechopen.com/books/images_new/3092.jpg",editedByType:"Edited by",editors:[{id:"50017",title:"Prof.",name:"Sylvie",surname:"Manguin",slug:"sylvie-manguin",fullName:"Sylvie Manguin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"371",title:"Abiotic Stress in Plants",subtitle:"Mechanisms and Adaptations",isOpenForSubmission:!1,hash:"588466f487e307619849d72389178a74",slug:"abiotic-stress-in-plants-mechanisms-and-adaptations",bookSignature:"Arun Shanker and B. Venkateswarlu",coverURL:"https://cdn.intechopen.com/books/images_new/371.jpg",editedByType:"Edited by",editors:[{id:"58592",title:"Dr.",name:"Arun",surname:"Shanker",slug:"arun-shanker",fullName:"Arun Shanker"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"72",title:"Ionic Liquids",subtitle:"Theory, Properties, New Approaches",isOpenForSubmission:!1,hash:"d94ffa3cfa10505e3b1d676d46fcd3f5",slug:"ionic-liquids-theory-properties-new-approaches",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/72.jpg",editedByType:"Edited by",editors:[{id:"19816",title:"Prof.",name:"Alexander",surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"314",title:"Regenerative Medicine and Tissue Engineering",subtitle:"Cells and Biomaterials",isOpenForSubmission:!1,hash:"bb67e80e480c86bb8315458012d65686",slug:"regenerative-medicine-and-tissue-engineering-cells-and-biomaterials",bookSignature:"Daniel Eberli",coverURL:"https://cdn.intechopen.com/books/images_new/314.jpg",editedByType:"Edited by",editors:[{id:"6495",title:"Dr.",name:"Daniel",surname:"Eberli",slug:"daniel-eberli",fullName:"Daniel Eberli"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"57",title:"Physics and Applications of Graphene",subtitle:"Experiments",isOpenForSubmission:!1,hash:"0e6622a71cf4f02f45bfdd5691e1189a",slug:"physics-and-applications-of-graphene-experiments",bookSignature:"Sergey Mikhailov",coverURL:"https://cdn.intechopen.com/books/images_new/57.jpg",editedByType:"Edited by",editors:[{id:"16042",title:"Dr.",name:"Sergey",surname:"Mikhailov",slug:"sergey-mikhailov",fullName:"Sergey Mikhailov"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"1373",title:"Ionic Liquids",subtitle:"Applications and Perspectives",isOpenForSubmission:!1,hash:"5e9ae5ae9167cde4b344e499a792c41c",slug:"ionic-liquids-applications-and-perspectives",bookSignature:"Alexander Kokorin",coverURL:"https://cdn.intechopen.com/books/images_new/1373.jpg",editedByType:"Edited by",editors:[{id:"19816",title:"Prof.",name:"Alexander",surname:"Kokorin",slug:"alexander-kokorin",fullName:"Alexander Kokorin"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}},{type:"book",id:"2270",title:"Fourier Transform",subtitle:"Materials Analysis",isOpenForSubmission:!1,hash:"5e094b066da527193e878e160b4772af",slug:"fourier-transform-materials-analysis",bookSignature:"Salih Mohammed Salih",coverURL:"https://cdn.intechopen.com/books/images_new/2270.jpg",editedByType:"Edited by",editors:[{id:"111691",title:"Dr.Ing.",name:"Salih",surname:"Salih",slug:"salih-salih",fullName:"Salih Salih"}],productType:{id:"1",chapterContentType:"chapter",authoredCaption:"Edited by"}}]},chapter:{item:{type:"chapter",id:"70602",title:"Numerical Simulation of the Spin Coating of the Interior of Metal Beverage Cans",doi:"10.5772/intechopen.90381",slug:"numerical-simulation-of-the-spin-coating-of-the-interior-of-metal-beverage-cans",body:'When a liquid is applied to a spinning substrate, or if a pre-wetted substrate is spun, centrifugal forces act to drive any irregularities in the film thickness outward, away from the axis of rotation. The result is that the film becomes thinner and more uniform as the rotation proceeds. Consequently spin coating is used in such applications as coating magnetic storage discs, optical devices, and semiconductor wafers to obtain very thin but uniform films on flat substrates.
When the substrate is curved, centrifugal forces will act to produce a uniform layer only in horizontal regions where the substrate is perpendicular to the axis of rotation. But the coating layer may be very irregular in regions where the substrate is highly curved and the normal vector from the surface is not parallel to the axis of rotation.
In this work we will derive the lubrication form of the fluid mechanical equations for a thin liquid film sprayed on an arbitrarily curved, rotating, axisymmetric substrate. Our goal is to predict how the coating thickness changes with time as a function of the substrate geometry, the rotation rate, the rheological properties of the coating liquid, and the geometry and flux of the spray gun. We then discretize the equations and solve the partial differential equations governing the flow. Using an implicit method of solving the finite difference representation of the partial differential equations, we require a minimum of computer resources.
The theory we develop in this work is used to analyze one specific application: the spray/spin coating of the interior of aluminum beverage containers. When a spinning can is spray painted, centrifugal forces help to cause a more uniform coating layer on the can substrate. Indeed this is the purpose of rotating the can a high spin rates while spray coating the interior. But centrifugal forces can also cause such coating irregularities as drop formation. In this work we will demonstrate how such parameters as the spray gun placement and the rotation rate contribute to how long the beverage can may remain in the spin phase of its coating process before this potential defect occurs.
Consider an arbitrarily curved, axisymmetric substrate with the parameter s representing arc length along the substrate. The parameter
Curvilinear coordinate system. ϕ is the circumferential angle, es is the unit vector parallel to the substrate, en the unit vector perpendicular to the substrate, and rs is the distance from the centerline to a given point on the substrate. θ is the angle of the substrate with respect to the horizontal, ω is the rotation rate, and g is the acceleration of gravity.
Metric coefficient | Coordinate | Velocity |
---|---|---|
Coordinates and their metric coefficients.
If
Here
If
We define
At the free surface, the substantial derivative of F must be zero. Consequently the kinematic condition on the free surface is given by
Employing the substitution
the mean curvature of the free surface for this geometry is given by
at
If the atmospheric pressure is zero, then the tensor equation relating the change in pressure across the free surface due to surface tension is given by
Here
If we sum the three equations found in Eq. (5) over
all evaluated at the free surface
We now scale the dependent and independent variables with various characteristic lengths of the substrate geometry. These include
If
We can eliminate the pressure from the
The scaled form of the continuity equation is given by
At the substrate,
The scaled form of the kinematic condition is shown to be
at
at
Instead of transforming the equations representing the pressure discontinuity across the liquid interface, given by Eq. (9) and Eq. (10), we will eliminate the pressure term in Eqs. (9) and (10) using the momentum equations and the identities for the partial differentiation of implicit functions. Eq. (7) gives the pressure in the liquid at the free surface
Plugging the pressure at the interface, found by Eq. (7), into the momentum equations yields
where
Here the
We now expand the scaled velocities in a regular perturbation series expansion in powers of the small parameter
We then use boundary conditions to determine the appropriate constants of integration and solve for
where
with the constants
Using the appropriate boundary conditions, the order
The evolution equation, to order
The dimensional evolution equation is found to be given by
We shall employ the maximum radius of the substrate as our length scale,
Substituting these scale factors in the dimensional evolution equation, Eq. (22), leads to the nondimensional evolution equation
in nondimensional units. Here
The geometry of the substrate is delineated by a schematic drawing which gives us the radius of each circular arc,
Substrate made up of straight segments and circular arcs of radius Ri and subtended angle φi.
We assume that the coating layer is laid down over time by a spray gun that emits a fan of gas that is directed toward the can substrate. Thus the accumulation of coating with time due to this fan is a function of s and t:
Assumed spray pattern.
If
If the flux does not vary significantly in the
Beverage can and spray pattern geometry. Here υ is the distance from the orifice of the spray gun to a given point on the can substrate.
When expressed as a function of
From Figure 4 we have the relation
For a sufficiently thin fan, where the point on the substrate is sufficiently far from the centerline
When we are near the centerline, this formula must be modified to
to account for the fact that here the substrate is constantly being reached by the spray fan.
We also assume that there is a secondary “gas” which is uniform in the interior of the can and results in a constant
where
In an effort to determine the maximum value a droplet can form without detaching from the substrate, we will consider an axisymmetric droplet forming on the underside of a ceiling, as illustrated in Figure 5. Here
Profile view of axisymmetric pendant drop.
If we nondimensionalize the problem by scaling
Here
Using a standard Runge–Kutta method, we can numerically integrate Eq. (27) assuming
If
Approximately 400 billion two-piece, all-aluminum cans are produced annually for the purpose of storing beverages for distribution worldwide. The interior of each of these cans must be coated to protect the aluminum from onslaught due to corrosive elements in the contained beverage, and the beverage must be protected from picking up metal ions or other off-flavors from the aluminum substrate. Consequently the coating must be as uniform as possible for thick regions may slough off and thin regions may not offer adequate protection. To achieve a uniform film thickness, spin coating is employed using a spray fan to distribute the coating on the can substrate. But because the can is highly curved due to structural considerations, achieving a uniform final film thickness is much more complicated than for a flat substrate. In this section we will apply the analytical and numerical model we developed in previous sections to determine how the many parameters are influencing the flow of the paint coating. These parameters include the rotation rate, the shape of the can, the coating fluids physiochemical properties, and the geometry and flux of the spray fan, all affecting the final film thickness distribution.
The can body is initially punched from sheet aluminum and then goes through a washing process to produce a substrate suited for the spray coating. The can is then spun at between 2500 and 3500 rotations per minute, and one or two spray guns spray the interior with the liquid paint film. Centrifugal and gravitational forces redistribute this liquid layer as the can continues to spin after the initial spray process. The can is then placed in an oven where the solvent is allowed to evaporate leaving only the hardened resin. Then the can is filled with the beverage and the top of the can is attached in place. This conveyer process can produce as many as 1700 filled cans per minute.
In practice, one or two spray guns are used to coat the interior of the spinning cans. These are oriented at between 5° and 30° with respect to the vertical axis of the can and placed between 0.5 and 1.5 cm vertically from the top of the sidewall [6, 7]. Typically the can is sprayed for between 0.05 and 0.2 s and spun for an additional 0.1–0.5 s [6] so that centrifugal forces can act to redistribute the coating layer.
The industry uses schematic drawings which plot the substrate as a function of circular arcs of radius Ri, and subtended angles
Profile of the can substrate in red lines and the coating layer in blue lines at t̂=.10, in the middle of fast spin with spray phase. Dimensionless variables are used.
Profile of the can substrate in red lines and coating layer in blue at t̂=.20, in the middle of the fast spin phase. Dimensionless variables are used.
Profile of the can substrate in red lines and coating layer in blue at t̂=t̂max=.45, near the end of the fast spin phase. Dimensionless variables are used.
We will assume that the spray fan has an elliptical cross section with a ratio of the major axis to the length of the minor axis of 10. The parameters determining the placement and orientation of the spray gun are illustrated in Figure 9. For the simulation considered in this work, the nondimensional parameters are listed in Table 1, the dimensional parameters in Table 2, and typical properties of the coating liquid in Table 3.
Profile view of can and spray gun placement and orientation parameters.
Parameter | Symbol | Value |
---|---|---|
Distance of spray gun from centerline: fan #1, fan #2 | −0.15, −0.45 | |
Distance of spray gun above can: fan #1, fan #2 | 0.15, 0.45 | |
Angle of spray gun wrt vertical: fan #1, fan #2 | 28°, 15° | |
Subtended angle of spray fan | 100° | |
Time spray gun acts in fast spin phase | 0.15 | |
Percent of secondary spray | 5% |
Nondimensional parameters for this simulation.
Parameter | Symbol | Value |
---|---|---|
Can radius | R | 3.33 cm |
Rotation rate | 2500–3500 RPM | |
Distance of spray gun from centerline: fan #1, fan #2 | A | −0.5 cm, −1.5 cm |
Distance of spray gun above can: fan #1, fan #2 | B | 0.5 cm, 1.5 cm |
Average wet coating thickness | 0.0028 cm | |
Time spray gun acts in fast spin phase | 0.05 s |
Dimensional parameters for this simulation.
From Eq. (26), we see that the coating applied by the spray gun is an inverse function of the radius of the can substrate
The dimensionless parameter
Critical time for droplet formation, t̂max, versus centrifugal force parameter. Here dimensionless variables are used.
The surface tension, density, and viscosity of the coating liquid are difficult to significantly alter as they depend on the required organic solvent content and surfactant levels in the paint formula. Similarly, R, the beverage can radius, is fixed by industry production standards. This leaves the rate of rotation as the only significant production parameter for changing the nondimensional (centrifugal force)/(surface tension force) parameter.
The centrifugal ejection of coating liquid from the inner wall of the moat is also predicted to be a strong function of the position and orientation of the spray gun. In the above example, the gun is placed 0.5 to the left of the centerline, is 0.5 cm above the top of the can, and is angled at
Critical time for droplet formation, t̂max, versus centrifugal force parameter for two different spray gun placements. For fan #1, the angle of inclination is 28∘, A1=−0.15, B1=0.45. For fan #2, the angle of inclination is 15∘, A2=−0.45, B2=0.45. Dimensionless variables are used.
In this work we have used scaling arguments and perturbation theory to derive the lubrication form of the governing fluid mechanical equations for a thin liquid film coating an arbitrarily curved, axisymmetric, rotating substrate. Our main purpose has been to develop mathematical model that can be employed to numerically simulate the application of a paint film to the interior of beverage cans, though the basic algorithm may be useful in other applications. We have used our algorithm to predict the time of centrifugal ejection of coating from the inner moat wall as a function of several input parameters: the physiochemical properties of the coating liquid, the rotation rate, and the spray gun placement. The model can also be used to predict other coating defects and how the input parameters can be used to avoid them. The effect of solvent evaporation during the drying phase, when gravity and surface tension forces affect the coating distribution as the viscosity increases until only a final, hard, film remains, may also be modeled. With so many parameters regulating the final coating thickness, using experiments to model the coating evolution and measure the final, dry, film thickness is an almost impossible task. Instead we can utilize the power and versatility of computer simulation to predict the coating profile as a function of input parameters. This understanding will be useful in optimizing the current application process. It may also be essential in acquiring a satisfactory coating when environmental regulations require a change to high solids and latex paints.
Bacteriophages, often shortened to just phages, are viruses that infect bacteria. Their discovery and characterisation in the early days of bacterial molecular biology has led to certain phages being very well understood in terms of their life cycle, and several phages that infect Escherichia coli have become tools in molecular biology techniques such as cloning [1, 2, 3]. There has been a resurgence recently in the use of bacteriophages as therapeutics, as vectors for the delivery of vaccines [4], for the killing of pathogenic bacteria as an alternative to antibiotics [5] and for gene therapy to transfer DNA to target human or animal cells [6]. Some of these uses would need the production of many millions of doses of a vaccine, for example, or very large quantities for use as an antibacterial. This has increased demand for investigation into the large-scale production of bacteriophage which would necessitate volumes from hundreds to thousands of litres. The use of phage as biotherapeutics such as vaccines or for gene therapy may be advantageous as phage is considered cheap to manufacture, with large quantities of the product being rapidly produced. But the large-scale production of wild type or genetically modified bacteriophages for use in the biotherapeutics industry provides significant process and regulatory challenges. Bacteriophages, like any virus, are dependent on a host organism to propagate, in the examples here it is E. coli; consequently, the generation of progeny bacteriophage is unequivocally linked to the physiology, molecular biology and growth needs of the host which are important to understand in order to maximise production.
\nMethods for the production of phages, e.g. λ and M13 bacteriophage, at laboratory production scale have remained unchanged for many years [7]. However, aspects of these protocols are either not practical or unsuitable for large-scale production of phages. Therefore, it is highly desirable to consider early on in the development of phage technologies how any successful bacteriophage therapeutic would be produced at large scale at an industrial level.
\nOne of the problems associated with producing and using λ as a biotherapeutic is the issue of host-derived nucleic acid. The λ lifecycle [8] involves the phage progeny escaping from the host cell by lysis of the bacteriophage host, whereupon the cell contents including high-molecular-weight host chromosomal DNA and RNA are released into the culture supernatant. This significant quantity of host cell-derived nucleic acid can cause important problems for both downstream processing [9] and from a regulatory point of view [10], so reducing the presence of bacterial host nucleic acid in the first stages of the process stream would remove these issues.
\nM13 is an unusual phage because it does not lyse its host and the entire phage is secreted from the host bacterium through special pores spanning the cell wall [11] although this does make the culture supernatant relatively free of contaminating host cell material, unlike the supernatant in a λ fermentation. In both lytic and secreted phage production the first downstream stage is the concentration of the phage from whatever volume of growth medium was used to grow the infected cells. Filamentous phage such as M13 has a very asymmetric shape with wild-type M13 having a length to width ratio of 138:1 and this extreme asymmetry allows a mild precipitation using polyethylene glycol (PEG) [12].
\nIn this paper we present initial studies into the parameters that need to be manipulated for scaling up fermentation of M13 phage for industrial production.
\nLambda (λ) bacteriophage is a temperate phage with a double-stranded (ds) genome of approximately 48 kb [13]. This is encapsulated in an icosahedral capsid (~50 nm in diameter) with a long flexible non-contractile tail (~150 nm in length). The host for λ production is E. coli with infection by lambda phage taking place via the maltose binding protein, LamB. Lambda bacteriophage is one of the most intensely studied bacteriophages and has been used for many studies on uncovering basic molecular biology [14] and in biotechnology for phage display of peptides and proteins [15], vaccine [16] and gene transfer and therapy [6].
\nThe large-scale production of genetically modified lambda bacteriophage for use in the biotherapeutics industry provides significant process and regulatory challenges.
\nOne of the problems associated with producing and using lambda bacteriophage as a biotherapeutic is the issue of host-derived nucleic acid. The lambda lifecycle involves the cell lysis of the bacteriophage host, whereupon high-molecular-weight host chromosomal DNA is liberated into the culture supernatant. The presence of large quantities of host cell-derived nucleic acid can cause significant problems during processing as high-molecular-weight chromosomal DNA causes an increase in the cell lysate viscosity [9]. Furthermore, the presence of nucleic acid in the final product is non-desirable from a regulatory perspective [10], and thus reducing its presence in the first stages of the process stream would alleviate these issues. The lysis of the host E. coli cell and the release of intracellular contents (DNA, RNA and proteins) as well as fragments of cell wall will have detrimental effects when processing lambda. For example, intracellular contents can co-precipitate with the phage, can compete for binding sites on chromatography material and can block membranes and chromatography columns. These contaminants need to be taken into consideration when planning a large-scale purification and downstream processing protocol.
\nM13 is an unusual phage as it has a filamentous structure of 900 nm in length and 6.5 nm in width. It is a member of a small group of closely related phages including F1 and Fd [17] that infect E. coli. The genome is a single-stranded circular DNA molecule, and the length of the phage (but not its width) is simply determined by the size of the page genome. Short phage particles can be made using plasmids that contain just the replication origin and packaging signals, and phage particles longer than the wild type can be made by inserting DNA sequences into the phage genome. It was thought that the very long but thin shape of M13 and other filamentous phages would increase their shear sensitivity in the various kinds of industrial-scale processing equipment of pumps, continuous centrifuges and membrane filters. This was seen not to be the case [18] which is highly advantageous for large-scale downstream processing of this and other filamentous phages. Filamentous phages have a rather special property in that they do not lyse their host, but set up a permanently infected state, and new, progeny phage is extruded through special structures in the cell wall. Derivatives of M13 phage were extensively used in the early years of DNA sequencing by the Sanger method [19] and in the techniques of site-directed mutagenesis [20] and phage display [21] for the maturation of recombinant antibodies [22].
\nThe unusual mode of growth of filamentous phage by secretion from the host without lysis has considerable advantages for these molecular biology methods because the phage in the supernatant of growing cells is relatively free of any cellular contaminants [23] such as intracellular proteins, genomic DNA and RNA. This makes the purification of filamentous phage a relatively simple matter with many fewer contaminants than phage λ cultures.
\nThe multiplicity of infection or MOI is the number of phage particles added per host cell to initiate infection and thus production. The methods developed for the uses of phage λ and M13 at small scale or a few mL generally tend to use a high MOI of 5–10 or more. The MOI is important for scale-up as it defines how large the culture that provides the phage for the scale-up needs to be. It is neither desirable nor sensible to have to grow a fermenter of phage to inoculate a slightly larger fermenter in the final preparation. Also with a large MOI of just under 10, we reach the point where there will be enough phage for every cell to be infected, and at that MOI we can only expect a single burst of phage particles for lytic phage and therefore only a small increase in the number of phage added. With an MOI of 1, only 63% of cells will be infected by one or more phages because there is a Poisson distribution of MOI to infected cells [24]. But some phages have many binding sites per cell, e.g. T4 has 105 molecules of OmpC to bind to [25], while M13 only has approximately 3 [26], so the kinetics of phage finding and attaching to bacteria and forming a productive infection are quite complex. There are 30,000 LamB proteins in the outer membrane which is the initial receptor for λ but many fewer copies of the mannose receptor, ManY, in the inner membrane which is where the DNA of λ crosses the inner membrane [27, 28]. For M13 the receptor is the tip of the F plasmid’s transfer pilus, and there are usually one or two F pili per cell. But once M13 has established its replication inside an E. coli host, the cell is then permanently infected and will continue to secrete phage from these intact cells.
\nUnder ideal conditions the burst size of λ phage particles is 170 +/− 10 which takes 51 min [29], and during this time uninfected cells will still be growing and dividing, providing new hosts for the phage that are released. To get repeated rounds of replication, the ideal cell numbers and MOI for each phage are different and take into account cell division rates, numbers of receptors, the choice between a lytic and lysogenic cycle in phages where those can take place, the burst size and rate of replication and maturation of the phage. This complex interaction of several parameters means that it is difficult to say a priori what the combination of cells, phage and time of addition is the most appropriate for a given size of growth chamber.
\nFigure 1 shows the relationship between the host E. coli and MOI of M13.
\nTitre of M13 produced from different MOI on E. coli TOP10 F′. M13 phage was prepared from a 400 mL culture of E. coli TOP10 F′ and precipitated with PEG 6000 at a final concentration of 3.3% and 330 mM NaCl. The precipitated phage was centrifuged at 14,000 ×g for 10 min at 4°C and resuspended in 8 mL of 10 mM Tris.HCl pH 7.5 and filtered through a 0.22 μm filter. The stock of M13 was approximately 1 × 1012 pfu/mL. E. coli TOP10 F′ was grown in 400 mL of Nutrient Broth No. 2 (Oxoid) and a 40-mL inoculum in 2-L shake flask at 37°C. Around 2 mL of M13 was added at the appropriate dilutions to achieve the three different MOI of 0.00005:1, 0.05:1 and 50:1. Each point is the average of three flasks.
The graph of MOI and final phage production in Figure 1 shows that from a 106 range of MOI added at the start of growth in Figure 1, all three cultures reach the same final M13 phage titre. It just takes slightly longer for the lower MOI cultures to reach the final of approx. 5 × 1011 phage per mL. This has important consequences for scale-up of M13 production. For example, if a large fermenter of, say, 100 L were needed and the MOI of 50:1 was needed, we would need to have 500 mL of the equivalent inoculum used here. The information from Figure 1 shows that we can use just 0.5 mL of the same titre inoculum or much less, e.g. down to a few microlitres. For convenience it is best to inoculate a fermenter with enough in terms of volume that will reach the medium in the fermenter, so a few millilitres of a phage dilution are all that is needed. This means that one phage stock can be used for multiple fermentation runs.
\nThe standard way of initiating infection of λ or M13 is to mix the phage and cells and allow a static incubation for usually 5–15 min for phage attachment to the phage binding target on the cell surface. The phage/cell mixture is then added to broth if liquid growth is desired or to 3 mL of soft agar and poured onto an agar plate if plaques are wanted. This static incubation is in the recipes for all phages being handled at the lab scale and probably came about because researchers thought it would maximise the attachment of phage to their host. However, in a fermenter it is not sensible to turn off the stirrer and let the cells sit for 15 min while phage attachment takes place. We tested what would happen if M13 phage were simply added directly to shaking cultures of E. coli JM107 without stopping the shaking and comparing this with a static attachment/incubation of 30 min.
\nFigure 2 shows this experiment with three different inocula of M13 phage giving MOI ranging over a 106 range.
\nStatic versus shaking incubation of M13 with E. coli TOP10 F′. The conditions and culture quantities were identical to those shown in Figure 1 with the addition of three conditions at each MOI where the M13 phage was added and left for 30 min static incubation. These cultures for the static incubation were then grown with shaking at 200 rpm in a 37°C orbital incubator. Each point is the average of three flasks.
It is clear from Figure 2 that the static incubation is not needed for the initial attachment of M13 to sensitive E. coli, and so the required dose of M13 phage can be directly added to a fermenter with the E. coli host already growing with the impeller stirring. The culture which will be stirred at a high rate does not need to be stopped and left to go static for 15–30 min. This would compromise the growth of the cells in a large fermenter, and so our findings give a positive help for the scale-up of phage and the way one can run a large fermenter where procedures have to be different from what is done at small scale in the molecular biology lab.
\nThe above sections on MOI and removing the necessity for a static attachment of phage were shown with M13 as examples. The M13 phage does not lyse its host, so the culture medium after infection is uncontaminated by the bulk of the host cellular contents and largely contains just the filamentous M13 bacteriophage particles. We have examined the supernatants from M13-infected cultures and determined the levels of host cell contaminants of DNA and protein [23] which are quite low compared to the large amount of cellular DNA, RNA and protein released by lytic phage. A phage such as λ is a representative of lytic phage which is the majority of the types of phages used in therapy and biological control for the replacement of antibiotics. At each cycle of replication and release of the new phage particles, the host is lysed, and the cell contents are released into the medium along with insoluble debris from the cell wall and membrane. This leads to problems in subsequent downstream purification due to the large amount of different cellular molecules competing in the subsequent downstream processing steps.
\nThe host RNA and DNA represent major contaminants that need to be removed especially for gene therapy applications. The release of host cell DNA also increases the viscosity of the medium, and this has an adverse effect on clarification by centrifugation and membrane concentration due to blocking of the membranes. For lab-scale molecular biology work, it is normal to add the enzymes pancreatic RNAseI and pancreatic DNAseI from bovine pancreas preparations. With the advent of bovine spongiform encephalopathy (BSE) which peaked in the 1990s, the addition of any bovine or animal proteins into the growth or purification train of material destined for human therapy was banned. These regulatory restrictions removed the ability to use these cheap nucleic acid-degrading enzymes, and the substitutes from bacterial sources were much more expensive. A strategy to overcome this problem was developed by us, and this was to express a broad-spectrum nuclease in the periplasm of E. coli which would be released as cells were lysed [30, 31]. The enzyme Staphylococcal nuclease has been extensively characterised and used from the 1960s onwards [32] and can degrade both DNA and RNA. The expression of this in E. coli where it is secreted into the periplasm does not affect the growth of E. coli because the enzyme cannot get access to its substrate while the cell is growing normally. When the cells are lysed by a bacteriophage such as λ or by homogenisation, the nuclease can gain access to the released DNA and RNA and degrade them. This ‘cell engineering’ approach to assisting bioprocessing was developed at UCL and has been shown to give considerable gains in the centrifugation steps and other downstream purification steps in bioprocessing of proteins such as Fab fragments [33, 34, 35].
\nWe sought to apply this cell engineering strategy for the production of λ phage and to help solve the problem of the large amount of cellular contaminants released into the media when λ phage cultures need to be harvested and processed.
\nE. coli JM107 [3] was grown in 2 L fermenters with either JM107 or JM107 containing the plasmid pMMBompnucB which is a broad host-range plasmid vector based on an Inc. Q plasmid RSF1010 and contains the Staphylococcal nuclease which has been altered by the addition of the E. coli ompA signal sequence for secretion [31]. Figure 3 shows the growth of the two hosts with no added λ phage and the same hosts with 8 × 1010 λ phage particles added after 2 hours when the OD600 had reached 10.
\nGrowth of E. coli JM107 and λ production with and without a nuclease-expressing plasmid. E. coli JM107 containing pMMBompnucB and with no plasmid was grown in 2 L (working volume 1.5 L) LH 210 series fermenter (Bioprocess Engineering Services, Charing, Kent, UK) with 150 mL of E. coli inoculum and a final working volume of 1.5 L in phage media containing 100 μg/mL ampicillin and 20 μg/mL IPTG. λ phage particles were added at 2 hours to give an MOI of 0.05 (4 mL of 2 × 1010 pfu/mL). Impeller stirring continued throughout the growth and addition of phage, and the OD600 was monitored.
The addition of λ to the fermenter used the strategy that we had developed where a low MOI is used and without a static initial incubation of the host cells and the phage. In this way we can add the phage directly to a growing fermenter of host with the impeller (single shaft with three top-driven, equally spaced, six-bladed turbines) and four diametrically opposed baffles in the fermenter, running throughout. The growth profiles of the two uninfected cultures show no difference in their growth profiles which means that the presence of the expressed nuclease enzyme in the periplasm has no effect on growth rate or final OD. In the two cultures with added λ phage, the OD drop is the same for both hosts showing that λ replication and cell lysis are the same in both.
\nThe production of λ was monitored throughout growth and is shown in Figure 4.
\nProduction of λ phage in 2 L fermenters with and without periplasmic nuclease expression. λ phage was added at 2 hours in Figure 3, and the graph here shows λ phage amounts from the 2 hours onwards. Samples from the fermenters shown in Figure 3 were diluted in phage buffer and titred on E. coli JM107 using soft top agar and plaque counting.
Both the nuclease and the non-nuclease-expressing E. coli JM107 produced the same 5-log increase in phage particles with the same time profile. The efficacy of the nuclease in the removal of the host nucleic acid was assessed by the electrophoresis of samples from each time point on agarose gels and the visualisation of the released nucleic acid (both DNA and RNA). Figure 5 shows the complete degradation and removal of the released host genomic DNA and host RNA in the strain of E. coli expressing the periplasmic nuclease.
\nDegradation of host nucleic acid from l infected E. coli JM107 and JM107 containing pMMBompnucB. (A) Agarose gel with samples from fermenter in Figure 3 growing E. coli JM107 with λ. 1, 1 kb ladder; 2, 0 h; 3, 1 h; 4, 2 h; 5, 3 h; 6, 4 h; 7, 5 h; 8, 6 h; 9, 7 h; 10, 8 h; 11 λ HindIII ladder. (B) Agarose gel with samples from fermenter in Figure 3 growing E. coli JM107 containing pMMBompnucB infected with λ. 1, 1 kb ladder; 2, 0 h; 3, 1 h; 4, 2 h; 5, 3 h; 6, 4 h; 7, 5 h; 8, 6 h; 9, 7 h; 10, 8 h; 11 λ HindIII ladder. The same volume of sample from each fermenter time point was loaded onto each lane.
The presence of the expressed periplasmic nuclease is apparent from the difference in the samples in Figure 5B compared to the samples from the same time points in the fermenter with no expression plasmid for the Staphylococcal nuclease. Almost all of the released host genomic DNA and the large majority of the RNA has been degraded in the culture that expresses the nuclease. Figure 4 shows that the production of λ phage particles is identical in both fermenters, and the presence of the nuclease does not impinge on λ production and leads to a removal of the majority of the nucleic acid that would normally need the addition of bovine pancreatic DNAseI and RNAseA or more expensive bacterial equivalents such as Benzonase™ [36] to reduce the amount of nucleic acids. This cell engineering approach means that no animal-derived enzymes need be added, no costly commercial bacterial enzymes need be added and the engineered cells provide their own nuclease which degrades the nucleic acid in situ, so no additional time for incubation of any added enzyme is needed. Therefore a saving of both time and money is achieved via cell engineering for bioprocessing.
\nBacteriophages produced at any scale need to be concentrated by some method after the growth and production have taken place. The properties of phages allow some precipitation methods that are milder than conditions needed to precipitate host soluble protein or nucleic acid. Phages are large multicomponent entities usually many hundreds of times larger than a medium-sized soluble protein. Their asymmetric shape also enables mild precipitation methods. Polyethylene glycol (PEG) precipitation is a mild method of precipitating biological material and is very efficacious in the precipitation of large asymmetric material such as DNA or macromolecular assemblies, e.g. virus-like particles such as phages [37, 38]. The larger and more asymmetric the particle, the lower the amount of PEG is needed to precipitate the particle and leave behind other smaller soluble materials. An exploration of different average molecular weights of PEG from 600 to 20,000 for M13 precipitation showed that PEG 6000 and PEG 8000 combined the best properties of precipitation at low % concentration with lower viscosities than PEG 12,000 and PEG 20,000 [23] and 2% PEG 6000 with 330 mM NaCl gave >95% precipitation of M13. The relationship between PEG and NaCl is shown in Figure 6 where the increasing PEG molecular weight and PEG concentration with % of M13 recovered were investigated.
\nThe relationship between M13 recovery and PEG molecular weight and percentage PEG. All precipitations were carried out at 10 mL scale with additions of PEG to the final % concentration shown in the diagram with 4 × 1012 CsCl purified M13 and 330 mM NaCl. The mixtures were incubated on ice for 1 hour and centrifuged for 10 min at 8000 ×g at 4°C.
The diagram in Figure 6 shows the relationship between increasing chain length/molecular weight of PEG, the % of PEG and the amount of M13 phage recovered. Higher-molecular-weight PEG preparations can be used at low final percentages, but there is significant increase in the viscosity of high-molecular-weight PEG solutions in the stock solutions needed to add to the phage-containing solution, making PEG 20,000 very difficult to use. PEG 6000 and PEG 8000 achieve virtually the same precipitation profile as PEG 12,000 and the lower viscosity of stock solutions of PEG 6000 make this chain length the best for precipitation with low viscosity. A concentration of 2% w/v PEG 6000 is ideal with 330 mM NaCl [23]. It was discovered that the nutrient media commonly used for growth such as NB2 contains sufficient salt (Na, K and NH3 ions) that the added NaCl can be reduced to only 135 mM which would make a saving in materials and disposal costs in large-scale M13 phage precipitation.
\nThe strategies for the scale-up of phage growth and primary downstream purification are still in their infancy, but we have shown that there are significant gains to be made from the work described here. The amount of phage that needs to be added to large-scale growth volumes can be reduced by several orders of magnitude from what is common at the lab scale. Phage can be added directly to fermenters where rapidly stirring impellers are needed to maintain aeration and correct physiology of the host with no cessation of the stirring, and the phage will find their host and attach with no difficulty. The use of cells engineered to produce their own broad-spectrum periplasmic nuclease gives significant gains in the destruction of host DNA and RNA release on lysis, and this prevents the contamination of the phage with nucleic acid when the phages are concentrated by precipitation. The gentle precipitation method of using low concentrations of PEG can then be used to give relatively pure preparations of phage in one step. These methods can be used together and will allow the large-scale uses of phage in the future in medical and clinical applications and then beyond into biotechnological applications such as the uses of filamentous phage in electronics like phage batteries [39] and the phage laser [40].
\nWe acknowledge and thank the Engineering and Physical Sciences Research Council (EPSRC) for support via the Life Science IMRC for Bioprocessing and the EPSRC for the PhD studentship to SB. We thank the Biotechnology and Biological Sciences Research Council (BBSRC) for grant funding to JMW and EKM to support ES under research grant BBD521465/1.
\nThere are no conflicts of interest.
.
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