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Optim User Manual v1.5.4

1. 122 APPENDICES cassscssccsiscusssoracssertasentnnnerietnehiseateobaceseechsccsaencaeuieesssceessnaneisenwoustennacontes 129 XVII PERA ES MAE e EEE E EE E EA EA E TEE 129 FVII CR OFER e E EE EAE A ENEE A 136 Page 4 Avacta age ANALYTICAL Avacta Analytical Ltd 2012 HOW TO USE THIS MANUAL Welcome to the Optim 1000 Installation and Operation Manual This section believe it or not is an instruction manual for your instruction manual There are some useful pointers here so don t despair Take a minute to read this and the rest of the document will be easier to interpret Software Instructions Avacta s software provides the human machine interface You will soon learn to navigate it by instinct but until you find your feet some things to look out for in the software have been highlighted for you using the following code Bold words indicate a tab or menu Italics indicate a Menu Option CAPITALS indicate a BUTTON e Direct instructions to the user are bullet pointed for clarity so you can scan the text and skip over the explanations if you wish PH This symbol is used to inform the user of a shortcut or toolbar button Glossary At the end of the guide there is a Glossary of Terms which explains all of the jargon that appears in this guide Throughout the guide text a word that has a specific meaning for the Optim 1000 software for example a tab heading Is indicated by a capital letter at the start f
2. report copied and exported in the usual manner Generating a sample report The Optim Analysis v1 5 software can be used to prepare a report that the user is able to edit save load and export Choosing options from the Analysis Type Dataset and Data Type will allow tables to be created containing the results of the tertiary analysis These can be added to the To access the report editor go to the Report Generator tab in advanced mode A report will be viewable with the title Optim Analysis Report that has a header that shows the name of the current study Avacta Analytical Ltd 2012 Avacta ANALYTICAL Page 111 THE OPTIM ANALYSER HANDBOOK AOOGAUNVH ais m O U gt Z gt lee lt n m re D El Optim Advanced Data Analysis Engine Beta Development Aya cta Manager Advanced Analysis Centre Report Generator ANALYTICAL Report MyNewReport UNSAVED Optim Analysis Report Study Data Within the report generator there are a number of buttons ADD NEW REPORT will create a new report that is by default called MyNewrReport There are no limits on the number of reports within the study RENAME REPORT will rename the current report OPEN EXISTING STUDY REPORT will load a report from the study folder that has previously been saved GSAVE REPORT INTO STUDY will save a copy of the currently open report into the study folder
3. x AuxlnoutData MatchMethod Method used to match x axis P 124 Avacta age ANALYTICAL Avacta Analytical Ltd 2012 This function performs the given mathematical function on the two input datasets The x values of the OufputData are the same as InoutData The AuxinoutData s x scale is adjusted to match those of the InoutData so that the point by point maths can be performed The Match Method options are Interoolate where the shape of the data Is preserved and Preserve Area where the integral of the data is preserved an example of this is to preserve photon counts in a soectrum when changing the x scale Interpolate InoutData Spectrum to be acted upon OutputData Resultant Start Value of first point or Auto End Value of last point or Auto NumPoints Number of data points or Auto This overrides Spacing Spacing Spacing between data points or Auto Rounding Round Auto parameters to nice values This function produces an interpolated version of the InoutData where the x scale is adjusted to be a uniform spacing The various options allow the user to control the details of the x scale limits and spacing FindMinMaxLoc InoutData Spectrum to be acted upon MinOrMax Whether looking for minimum or maximum MinMaxName Name of Results File This function gives as the output the x value corresponding to the maximum or minimum y value QZ LU Y gt i lt Z l
4. Avacta Analytical Ltd 2012 XVI EDITING AND DEVELOPMENT OF ANALYSIS METHODS One of the major new features of the analysis software v 1 5 is the ability to adapt and create new analytical methods to provide an optimal measure of the behaviour of measured samples The default methods The default methods are typically geared towards the unfolding and aggregation of protein samples containing tryptophan Primary analysis methods The default primary analysis methods consist of a method to analyse the static light scattering intensity called ScatterMethod and a method to analyse characteristics of the intrinsic fluorescence IntrinsicFluoMethod e Intrinsic Fluorescence o Calculates the integrated intensity of each spectrum in the wavelength range 280 460 nm The result of which is called Fluolntensity o Determines an intensity ratio between the spectral intensity at 350 nm to that at 330 nm called Ratio350_330 o Evaluates the expectation wavelength barycentric mean of the spectra in the range of 280 460 nm The barycentric mean is effectively the centre of mass or a weighted average of the fluorescence emission and is given by A dia fxa da f called BarycenFluo o Determines a peak position and a peak height by fitting a Lorentzian function to the fluorescence initially centred at 340 nm with a width of 20 points and performing five iterations called FluoPeakPos and FluoPeakHeight respectively
5. Page 1 Avacta J ANALYTICAL Avacta Analytical Ltd 2012 a 8 m U gt re gt Z General Instrument Safety The Optim 1000 was designed with due consideration of the Low Voltage Electromagnetic Compatibility and Machinery Directives 2006 95 EC 2004 108 EC and 98 37 EC respectively It has been independently tested to EN61010 1 2001 EN61010 2 81 2002 EN 61326 1 2006 EN55022 2006 A1 2007 EN5502 1998 A1 2001 A2 2003 EN61000 3 2 2006 EN61000 3 2 2006 EMC FCC Title 47 of the CFR 2008 Part 15 b EMC requirements Note This equipment has been tested and found to comply with the limits for a Class A digital device pursuant to part 15 of the FCC Rules These limits are designed to provide reasonable protection against harmful interference when the equipment is operated in a commercial environment This equipment generates uses and can radiate radio frequency energy and if not installed and used in accordance with the instruction manual may cause harmful interference to radio communications Operation of this equipment in a residential area is likely fo cause harmful Interference in which case the user will be required to correct the interference at his own expense Please be aware that any changes or modifications not expressly approved by the party responsible for compliance could void the user s authority to operate the equipment FCC CFR 47 section 15 21 Moving and lifting the Instrument
6. 48 Optim Advanced Data Analysis Engine Beta Development Av ac Manager Advanced Analysis Centre Report Generator Primary Secondary Tertiary 350 f Edit 300 3 nts nm x10 N ul t Scatter266 cou Page 120 Avactad ANALYTICAL Avacta Analytical Ltd 2012 The method that you are testing Is displayed in this area for reference purposes only and cannot be edited from this tab The data displayed in the preview area will be the selected data in the relevant analysis sub tab See above for instructions on how to select the test data To test the method against a different soectrum or curve in the collection click either or gt Click the RUN button to test the method and display the resulting datasets chosen by the checkbox in the Results dialog Calculated values that are output from the analysis method are displayed in the results box Results Characteristic Value std Dey Units Fluolntensity 1 1262e 06 counts nm Ratio350_330 0 91193 BarycenFlua 345 14 nm 1 FluoPeakPos 337 03 0 073125 nm I FluoPeakHeight 16074 285 47 counts In this case datasets DO the original data and D1 the background subtracted data are available and displayed The calculated results are shown with units and an uncertainty if available With both DO and D1 checkboxes ticked the Preview area displays both traces 60x10 QZ LU Y gt i lt Z lt oO O LU T
7. Taking a Measurement The chosen Experiment will have default settings for the measurements that the instrument will perform for example UV fluorescence There is no need to change any settings at this stage as the defaults have been chosen to be suitable for most applications Measurements View the Measurements tab to see which measurements will be performed Avacta Analytical Ltd 2012 OAyacta Page 47 Deep UV Intrinsic Fluorescence amp Blue Laser Scattering O Single Shot GC Start Run oO Abort Run File Plate Plate Stage i MCA 1 2 3 MCA 1 MCA 2 MCA 3 G Edit B Apply B Cancel aE gt Z U wo O O 7N INAWNALSNI ANY FAV MAOS LNANO If you wish to change the default exposure time slit width or centre wavelength e In the Measurement Settings window click BJ EDIT e Type in the required exposure time slit width and centre wavelength e Click EJ APPLY to apply the changes It could take several seconds to apply the changes Temperature Control The Temperature Control window displays the temperature settings that have been chosen for this Experiment Status This tab displays the temperature hold time and step count status throughout a Run Ramp Settings For a thermal ramp type Experiment the temperature will start at the Start Temperature and a measurement will be taken for each well of the MCA confi
8. Q i A unique high throughput micro volume protein analysis and characterisation system User manual v1 5 4 Avacta ANALYTICAL Avacta Analytical Optim 1000 v1 5 Issued By Avacta Analytical Ltd 651 Street 5 Thorp Arch Estate LS23 7FZ UK 44 0 844 414 0452 2012 Avacta Analytical Ltd All contents of this document are the property of Avacta Analytical Ltd This document is the confidential work product of Avacta Analytical Ltd and no portion of this document may be copied published performed or redistributed without the express written authority of Avacta Avacta Analytical Ltd 2012 P i n Ayacta CONTENT Avacta Analytical Optim 1000 V1 5 cceccssccesccesccecccsccesccesccescceccesceesceescees 1 CONTENE oneen E E A E 3 HOW TO USE THIS MANUAL eneeeeseseoeososessosososessososossososossosososessososossososessosososessos 5 PREPARA NON cociros EE 7 UE PIE PI TON ea E tote E EE EE E 7 System CSOD OMS ial sessessssesseseesesseseesessesersessesersessesesseserseeeesesseseesesseseeseseeseeseseeseee 7 Dimensions and weights sessesssesessesseesserersseserseesersseseesseseeseesersessesserserseseesseseeeeens 7 Environmental REQUIFEMENTS ccccccesssccceseeccceccuscesccusecsceusecsseuscceseusceeseusceeseuseeesees 8 Electrical ReguiremMentS eesseseesessesessessesersessesesscsersesseserseesesesseseeseseeserseseeseeseseesees 8 Eaa I Siac T E S E I E E EA
9. What further action is necessary By whom When User If the X Y stage moves when the user has their hand in the way The X Y stage should not move when the door is open This will prevent this from happening The stage moves slowly maximum rate 1cm s that the user would have sufficient time to remove their hand fingers before significant harm could be done if the stage were to move with the door open and the user has their hand inside The risk of this happening is therefore very low No further action is necessary as the risk is very low Avacta Analytical Ltd 2012 OAyacta Page 130 ANALYTICAL 3 Operation without side panels L DANGER Electrical Shock Hazard High voltage contacts may be exposed when the instrument panels are not in place Who might be harmed and how What is being done already What further action is necessary By whom When User Severe electric shock can result from operating the instrument without its panels in place by touching electrical terminals inside the instrument All electrical terminals can only be accessed by removing the instrument panels The user is advised to not remove the panels Severe electric shock can result from operating the instrument without its panels in place Do not remove the instrument panels Correct operation of the instrument is to only operate it when the panels are in place With correct operation the risk is very lo
10. e Scattering Intensity o Calculates the integrated intensity over the wavelength range 261 271 nm called Scatter266 o Calculates the integrated intensity over the range 468 478 nm called Scatter473 QZ LU Y gt i lt Z lt Q LU ale Vat B O1 S There are also two further methods which are called ScatterMethod_BufferSub and IntrinsicFluoMethod_BufferSub These provide the same functionality as the other two methods but include an additional step where the software will match the provided characteristic information about individual sample wells to find an appropriate spectrum to subtract For this fo function correctly you need to e Have entered identical formulation information in the well in which the buffer will be loaded but leave the Sample Details column empty e Have changed the reference column entry to TRUE Page 11 Avacta e ANALYTICAL Avacta Analytical Ltd 2012 AOOGAUNVH 4 EIE m Q gt Z gt i lt n m 0 This will generate output results which have an appended _B For example Ratio350_330 will be called Ratio350_330_B when a buffer soectrum has been subtracted The default methods in the analysis wizard are applied to the following default collections Spectra Collections Analysis Methods Buffer Subtraction Buffer Subtraction ON OFF ScatterMethod SampleNoBufferSpectra SampleSpectra INtri
11. gt Z U wo O O 7N INAWNALSNI ANY FAV MAOS LNANO The status bar at the bottom of the window displays the following information O The user that is currently logged on Othe Project that is currently in use The selected Experiment D The ID and title of the active Run ES The MCA configuration number of wells in the current Run UD Live status Updates about the Optim 1000 instrument and a progress bar File system Database The Optim software has an inbuilt database system that stores all newly acquired data sorted by Project Experiment and Run Within the software data can be loaded by the Run ID and viewed and all the results generated will automatically be saved to the database Exporting and Importing Data can also be exported from the program Any raw data calibrated data or analysis results will be exported in the Excel format Exported files will appear in a list in the Results tab and can be accessed in the folder C Optim Client Export Femi It is advisable to save exported files immediately in a different file location as the Export folder will be cleared each time the software is restarted P Avacta a ANALYTICAL Avacta Analytical Ltd 2012 Formulation details for the MCAs can be imported from Excel into the database The easiest way to do this is fo use the Edit Formulations file that appears as a shortcut on the desktop to generate formulation information It is also possible to
12. ANALYTICAL Avacta Analytical Ltd 2012 Choose either Analyst or Supervisor from the drop down list Click SAVE In this window e Click SAVE to save all the changes on this page Listed users can be edited deleted or disabled The Administrator will always be able to see a full list of the users and make changes to the list Deleting or Disabling User Profiles On the occasion that you want to remove a user from the database there are two options to disable or delete the user e Disabling a User To maintain access to the user s details but stop them trom logging in simply disable the login for that user by ticking the Locked box on their profile Their name will still appear in the list of Users but their login details will be disabled so that an error message appears if the user tries to log in ac gt Z U W O O 7N INAWNALSNI ANY AAV MAOS LNANO e Deleting a User Highlight the user s name in the list and click DELETE This will deactivate the user so that their name is removed from the list of Users and you will no longer be able to access their profile Any data that the user collected will still be accessible by other users through the Project database P 4 Avacta des ANALYTICAL Avacta Analytical Ltd 2012 X DATA MANAGEMENT In the Optim Client software there are two types of data Global and Project specific Most of the data that an Analyst level user encounters is Project spe c
13. EDIT e In the Formulation Details box fill in the following information for each well that contains a sample Sample Details Sample Reference pH Replicate No Sample Name Sample Conc Notes mg ml Buffer Details Buffer Conc M Additives Concentration Additive Additive Conc M g 5 y S E Cancel Avacta Analytical Ltd 2012 Page 41 Avacta ANALYTICAL CLIENT SOFTWARE AND INSTRUMENT HANDBOOK AE gt Z U W O O 7N INAWNALSNI ANY FAV MAOS LNANO e Standard Data o Well Sample Name Write a brief description of the formulation in the selected well This will be displayed in the list of acquired spectra and results o Well Sample Ref This reference is for the user only and should be chosen according to the user s company policy o Sample Select a sample molecule from the drop down list and enter the concentration in mg ml o Buffer Select a buffer from the drop down list and enter the concentration Molar M Administrators can add to the list of buffers via the Samples tab For instructions see section X on page 55 e Optional Data o pH Type in the pH for this formulation o Replicate No Keep a record of duplicate formulations by labelling them with the same replicate number o Additives To add an additive to the formulation click NEW select an additive from the drop down list and type in the concentration Molar M Click SAVE The additive will now b
14. EXPORT TO RTF will export the currently open report into the study folder as a rich text format for editing in a word processing package of your choice Adding content to report Within the analysis screens you will offen come across a button that adds data to the report By clicking on this button the graph will be added to the report under a relevant section heading The same can also be done with the results tables The report has a default style that constrains the graphs into categories of primary analysis secondary analysis and tertiary analysis As a result graphs from the relevant sections go into each section of the report The report is editable however and content can be moved around or deleted and text can be added Fume YOU MUST SAVE THE REPORT AFTER YOU HAVE MANUALLY EDITED IT BY CLICKING SAVE REPORT INTO STUDY OTHERWISE YOU WILL LOSE ANY TEXTUAL CHANGES YOU HAVE MADE IF YOU ADD FURTHER CONTENT THROUGH THE ANALYSIS WIZARD OR ADVANCED ANALYSIS MODE P 112 Avacta age ANALYTICAL Avacta Analytical Ltd 2012 Exporting report By clicking on S EXPORT TO RTF a Rich Text Format variant of the report will be exported to the Studies folder This can then be imported into Microsoft Word or document processing package of preference for further editing or importing into user standard templates QZ TE 7 gt lt Z lt 2 oO O Lu ale HANDBOOK P 11 Avacta age ile ANALYTICAL
15. O O wal QO Z lt IE aE gt Z U Ss o AN INAWNALSNI ANY FAV MEOS LNAMO 2 Place the black cuvette holder with pre loaded cuvettes into the groove on the Filling Tool so that it is held in place by the magnets 3 Use a single or 16 channel micropipette with an appropriate volume range to collect the sample formulations from the pre loaded row in the microtitre plate or sample vessel Item no 1022 4 Align the pipette tips with the micro L LA cuvettes using the grooves in the Filling ZF Tool indicated by an arrow in the ET illustration 5 Gently press the plunger ensuring that the formulations flow into the micro cuvettes 6 Place one seal in the correct orientation into each side of the blue frame These will seal the ends of the cuvettes and ensure that the formulations do not evaporate at high temperatures CROSS SECTION ul The round side of the seal should be in contact with the ends of the 1 ul cuvettes The square side is used to seal the ends of the 9 ul cuvettes Fa CROSS SECTION J ia 9 ul 7 Slide the frame onto the cuvette holder ensuring that the tongue and groove fittings mate correctly at each end of the black holder 8 Gently lift the MCA off the Filling tool and close the hinged part of the cap mating the tongue and groove near the clip Apply gentle pressure to compress the seals against the ends of the cuvettes and secure the
16. lt lt 3 283 2 2 2 222 2222 22222 nny TT eese m SOABS OUNY ejeg MEY NAY SNIMNG NN uol eINSIJUoD SUOIE NUWIOS oys ajus p suyas ysn pe ues asp te se ae snm s ajdwes E TENTENE ainjesedway NNY avOl sauljap sasn NOY MIN uolze4qi eD Sauljap Ajjed eWO Ny La JO ISl SULEUOD yuawn dx3 palo THa Jasn P Avacta ages ANALYTICAL Avacta Analytical Ltd 2012 VII QUICK START GUIDE Quick Start Outline Here is a brief summary of how to obtain stability information about your molecule of interest using the Optim 1000 Turn on Optim first by the switch at the back then the button at the front Switch on the PC Allow at least one hour for the laser in the instrument to warm up When all the lights at the front of Optim are no longer red open the Optim Client 1 5 software and Login to a Project Select an Experiment to start a new Run Decide the MCA configuration number of wells you wish to use and input the Formulation details for your Samples Fill the micro cuvettes with the formulations Open the door pull out the tray and load the Micro Cuvette Arrays MCAs onto the hot plate starting from the left Place the lid on the copper plate Push the tray in and Close the door View the Measurement details amend the temperature step settings and start the Run View the calibrated spectra in the View Spectra tab as they
17. lt E 1 Stepped Thermal Unfolding Study Aim To determine the formulation that provides the greatest resistance to thermally induced denaturation of proteins with increasing temperature Method Probing the intrinsic fluorescence and scattered UV light while stepping up the temperature of the sample The position and shape of the intrinsic fluorescence spectrum changes as the protein unfolds The sample is heated using a stepped profile All the samples are equilibrated at the measurement temperature and then held at this temperature while they are measured The temperature is then incremented the samples are equilibrated held and measured again 2 Isothermal Unfolding and Aggregation Study Aim To determine the formulation that provides the greatest resistance to thermally induced denaturation and aggregation of proteins at a constant elevated temperature over a period of time Method Examining the intrinsic fluorescence and UV and blue scattering as a function of time whilst the temperature is held at a constant elevated level When under thermal stress the proteins begin to unfold and aggregate This is indicated by an increase in the intensity of the scattered light and changes in the fluorescence spectra By using a UV laser for high sensitivity measurements and a higher power blue laser with a broader focus aggregate size differentiation can be achieved 3 Stepped Thermal Unfolding and Aggregation Study Aim To determi
18. 900123536 05 3 31 2010 20059 PM B1 2999 C 4074s 3 Jo o 299901685692002 60 45 503 5 5 00378737E 05_ 3 31 2010 2 01 04 PM c1 29 99 C 45 50s _ ja Jo f3o 29 9901685692002 60 50 237 5 497700457E 05_ 3 31 2010 2 01 08 PM D1 29 99 C 50 24s Is Jo o 30078795908962 6o 54956 5 5 0049417E 05 3 31 2010 2 01 13 PM E1 3008 C 5496s s Jo o 30078795908962 60 5933 5 488995975E 05_ 3 31 2010 2 01 18 PM F1 30 08 C 5933s_ z Jo fso 30078795908962 so esor9 f5 498531626 05_ 3 31 2010 2 01 22 PM G1 30 08 C 64 02s 8 Jo o 30078795908962 60 68704 5 495299209E 05_ 3 31 2010 2 01 27 PM H1 30 08 C 68 70s 9 Jo o 299901685692002 60 73435 5 493590633 E 05_ 3 31 2010 201 32 PM 1 2999 C 7344s 10 fo 3o 299015412294385 60 78093 5 5 04119125E 05 3 31 2010 2 01 36 PMI Ji 29 90 C 78 09s_ ja fo o 299901685692002 60 142511 5 487910838E 05_ 3 31 2010 2 02 41 PM K1 29 99 C 14251s a2 Jo 3o _ 299901685692002 60 147421 5 5 00817441E 05_ 3 31 2010 2 02 46 PM L1 2999 C 147 42s __ 13 Jo o 299901685692002 60 assis 5 495576314E 05_ 3 31 2010 2 02 50 PM M1 2999 C 15192s 14 Jo 3o _ 299901685692002 60 156 582 5 494352571E 05_ 3 31 2010 2 02 55 PM N1 29 99 C 156 58s a5 Jo o 299901685692002 60 161258 5 494929809E 05_ 3 31 2010 2 02 59 PM 01 2999 C 161 26s _ oOo P E ee 31 939070
19. Avacta Analytical Ltd 2012 Page 22 THE CLIENT SOFTWARE AND INSTRUMENT HANDBOOK The purpose of the Client software Handbook is to guide a user through the day to day operation of Optim 1000 Starting a New Experiment guides the Analyst user through every step of the settings and options that are involved in running a new experiment Sections in the analysis segment of this chapter introduce the user to loading and processing data after it has been acquired Some settings are hidden from the Analyst user and must be set up by the Administrator For instructions on those features that are exclusive to the Administrator and for in depth descriptions of the pre programmed software routines such as calibrations and analysis macros read the later sections that appear later in this handbook The purpose of these later sections is to guide an Administrator level user through the additional features of the software and to give further information about the default settings and options Here you will find detail about the calibration functions applied to the raw data the logic of the temperature control feature and some other useful information The administrator information is arranged into three sections User Management Data Management and Experiment Settings P 2 Avacta age ANALYTICAL Avacta Analytical Ltd 2012 a lt i 2 oS a O NN w Q Sy Ze OR SZ O O wal QO Z lt IE IV GETTING STARTED Th
20. BufferS pectra gt ScatterMethod_022 gt IntrinsicFluoMethod_022 ampleB ufferS pectra SampleN oBufferSpectra ScatterMethod_BufferSub SampleS pectra Analysis gt IntrinsicFluoMethod_BufferSut Program gt ScatterMethod_BufferSub v Perform Primary Analysis Performing primary analysis is simple in the advanced analysis mode A program is built up by the following process e Selecta collection e Select a method to use to analyse e Click ADD TO PRIMARY ANALYSIS PROGRAM e Repeat this process until you have built up your program e lf you make a mistake select the line in the Analysis Program that is wrong and click REMOVE FROM ANALYSIS PROGRAM e When you have completed building up your program click PERFORM PRIMARY ANALYSIS The example shown above creates an analysis program that will look at the light scattering and intrinsic fluorescence profiles of buffer soectra in order to determine what effect temperature has on their soectra and analyse the spectra by first subtracting a relevant buffer from each sample before calculating light scattering and intrinsic fluorescence characteristics Page 91 Avacta age ANALYTICAL Avacta Analytical Ltd 2012 QZ LU Y gt i lt Z lt oO LU ale Vat B O1 S AOOGAUNVH a8 m O gt Z gt P lt n m 7 The default primary analysis methods consist of a method to analyse
21. Clicking on 0 will copy the graph as an image and you can paste this into any software of your choice Right clicking on each axis will allow the scale to be changed Ticking AutoScale will automatically set the scale of the graph If this isn t ticked you can enter values for Min and Max as required Ticking Log Scale will display the axis as a log plot n Sea Re Scale Axis left E AutoScale Min 0 Max 10000 E Log Scale Cancel Test Apply P Avacta age ANALYTICAL Avacta Analytical Ltd 2012 Before setting the scale TEST will allow you to review the display before clicking APPLY to set the scale to the required values The button HE will autoscale the axes of the spectrum graph You can view the raw data as acquired on the CCD detector by clicking PLOT and selecting Raw Selecting Spectrum from this list will return to the normal calibrated spectrum view Performing primary analysis on spectra lists This section describes how to analyse the spectra collections using default analysis methods Creating and editing analysis methods is covered in a later section Go to Advanced Analysis Centre tab and the Primary sub tab and ensure that ANALYSE SPECTRA Is selected Avacta ANALYTICAL D Optim Advanced Data Analysis Engine Beta Development Manager Advanced Analysis Centre Report Generator Primary Secondary Tertiary Method Developer Analysis program
22. HANDBOOK 40 Intensity counts x10 sjunos Ayisuaju y Wavelength nm D ip f you expected to see more output datasets ensure that you have enabled the output for the dataset you want in the End function of the method Method management Methods that have been created in your study can be added to the default methods and vice versa so that they can be used in the wizard mode and in other studies Navigate to the Advanced Analysis Centre tab and the Method developer sub tab and ensure that ORGANISE is selected Page 121 Avacta a ANALYTICAL Avacta Analytical Ltd 2012 AOOGAUNVH 4 ais m O me lt gt Z gt P lt n m 0 Methods stored in current Study Primary ScatterMethod_022 IntrinsicFluoMethod_022 IntrinsicFluoMethod_BufferSub ScatterMethod_BufferSub azing_method0 Secondary l Tm_Method_022 Default Methods Primary ScatterMethod_022 IntrinsicFluoMethod_022 ScatterMethod_BufferSub IntrinsicFluoMethod_BufferSub ScatterMethod_01 Secondary Tm_Method_022 Tonset_Method_022 Tonset_Method_022 i Isothermal_Method_01 second derivative By selecting different methods that are in the study they can be added to the default methods by clicking COPY METHOD TO DEFAULTS By selecting a default method clicking COPY METHOD TO STUDY a default method can be added to the current study The default methods are present in all newly c
23. LUI Ae HANDBOOK Al B1 C1 E F1 Gl H1 11 11 K1 M1 NT 01 Pl Well 2 Markers the graph will be displayed with points indicating the value of the parameter Where there are multiple results they will all be overlaid The most significant result will have a larger marker TransTemp BarycenFlua Page 75 Avacta ANALYTICAL Avacta Analytical Ltd 2012 AOOGAUNVH 4 EIE m Q gt Z gt i lt n m 0 3 Table the data will be presented as a table showing the well reference sample description and the values of the parameter The colour of the bars and points that are displayed on the graph correspond to the key at the bottom of the graph If you wish to add or remove results from a particular well from the graph you can click the coloured square next to the well reference in the key to do so Clicking on one of the target buttons will toggle between hiding and displaying all wells in the selected MCA To export the secondary analysis result click EXPORT If you select All Data then the software will export all the primary analysis results If you select Displayed then it will export only the displayed graph data To add the secondary analysis results to the report click ADD TO REPORT Again by selecting All then all the data will be added to the report in the Secondary Analysis section By selecting Displayed then only the displayed graph will be added to
24. Ltd 2012 Collection of Selected Analyses Temperature BarycenFluo rmunUJ00005 417 61 07 07 E11 Temperature Scatterz66 rumnUO0005 41 61 01 01 E7 As before the selected collection can be renamed by clicking 4 deleted by clicking T and now exported to a csv file by clicking amp All exported data is saved to the study folder in the Studies location defined at the start of this handbook with a filename of ordinate_vs_abscissa csv If a file of this description already exists the new filenames will have a number appended sequentially with each export Clicking l will copy the displayed graph to the currently open report This is discussed later in this handbook The data in the graph can be quickly extracted for use in other programs by clicking COPY GRAPH DATA EXPORT GRAPH DATA will create a csv file in the study folder with a filename comprised of the ordinate names and the abscissa If a file of this description already exists the new filenames will have a number appended sequentially with each export QZ LU Y gt i lt Z lt Q LU ale Changing the view of the data for a clearer picture The button labelled w will allow you to smooth your data The data shown below is unsmoothed FluoPeakPos nm 20 40 60 80 100 Temperature C Despite this it is still easy to see the different trends in the samples in this case these samples are protein that has increasing concentrations
25. T A 9 Mice AFET ADVICE a truss eccees te seve sisson E A E odes taeetens 11 General Instrument Safety esesesseeeesesresresresresrrsrrsrrsrrsrrsresrrsreerteseseestesersreseeseeserseee 12 S169 26 EE a E E E E E E E 13 Bocs Eo elch A EE A A A E A E A E EA 13 PRAVICO OI OS e E R AE AE AE EE ENA 14 aaa i E E E OE nen EEE O N N PE 15 BOCI a AS aa E E AEE E EE E EE 15 PS OS I e E E E EEE EE 16 IIl SOFTWARE INSTALLATION AND ACTIVATION cccesccttectceccscenscettosnseseadssersye et aseaedtaeats 17 TOU SOT tate ha ear E E E E E EEE AEREE 17 Installing the Optim 1000 client sofware sseesesceereeresresresrrsresresresresresreseeerrerrereees 17 Installing the Optim Analysis SOTTWOIE cccccecccecccesceessusceusccusceescensceescesscesscesscees 21 ACtivating the analysis SOftWwWare ssessesseessssesseesseresresrerrersessesrerresresrersesrerrereersers 22 THE CLIENT SOFTWARE AND INSTRUMENT HANDBOOK cccccscsscevcscees 23 We COPR C TARE D E E E E E EEN 25 V POWERING OPTIM ON AND OFF esesessssrsrcssrrsrrreessersreseesrrsersrresersreeeersreseresns 27 TO TE N e E E R E EE 27 TST VCO OT e e E E EAE EEEE 27 TUA ESA E E AEE SE E E E EA i 28 Vi INTRODUCTION TO THE OPTIM CLIENT SOFTWARE ccccccccesccesescessseeesseeeseees 29 PVCU TIS Ss OWO O reen E E 29 Bl EEIE a TEE TEE TEE E EIEI IEO E T 30 WE EE E E EAE OANE EAN N N AE N IE TE 3 SHUI TOS OVEIO W seiere Tarain nTen EE AE E EEE AN 3 5 4 AUCE TARTE BD
26. TRUE m Click on a data to edt Selecting a study and a run within that study will allow the information to be edited In some cases where there are only a defined number of options available such as the Analyse column clicking on a cell in this column will present a popup menu containing the available choices Changes made here need to be applied so click APPLY CHANGES to save these changes back into the study ADD TO REPORT will add the information to the sample report as a series of tables containing related information that are formatted to fit into the width of the page P Avacta Se ANALYTICAL Avacta Analytical Ltd 2012 FP cea Changing the sample information has a direct impact on the content of the default tertiary analysis collections It is important to remake the default collections after changing the formulation information Only if you have changed information in order to perform a buffer subtraction will you need to remake the primary analysis collections Primary Analysis Primary analysis is the second phase of the data analysis process after data loading Analysis is completed by selecting a number of spectra forming them into collections and then applying an analysis method to a selected collection These collections can be either groups of related spectra so that different analysis methods can be applied as required or for simplicity s sake one collection containing all soectra
27. U W O O 7N Projects The Administrator can use this tab to create edit and delete projects in the database Supervisors can also create new projects in this tab The Projects tab contains a list of all projects that have been created Click on a Project to load all the settings associated with it or to edit the details INAWNALSNI ANY FAV MAOS LNANO Please select a project from the list below Bs Optim Demo New Q Edit T Delete To create a new Project Click Ga NEW Enter a title and description for the new Project The date that the Project was created should be filled in automatically Click G SAVE Go to the Users tab and choose the users and access levels for your Project Go to the Samples tab to create a record for each molecule you intend to study within this Project To view an existing Project Highlight the Project in the list on the left You will be able to see which user logged into this Project most recently and when the Project was created Click amp 4 EDIT to edit the details of the Project To view old data or add new data fo this Project go to the Experiments tab and LOAD a Run or create a NEW Run respectively To gain a better understanding of the data management structure within the software please refer to the flowchart Buffers and Additives The Administrator controls the global list of buffers and additives that are available to the user To add a new Buffer o
28. You can save these options by clicking SAVE SETTINGS in the General tab _ Save se ings Choosing the analysis mode you want to use With two wizard or advanced analysis modes built into the system making the correct choice for your needs is important to get right Luckily the analysis engine underneath is the same regardless of your choice so you can switch between them interchangeably at any time However when choosing how you wish to use the software it is useful to refer to the information below to ensure that you are appropriately informed and able to choose wisely Use the Analysis Wizard if you wish to Analyse data from a variety of samples within a single run using the same analysis method Obtain unfolding and aggregation curves versus either temperature or time depending on the experiment type you are doing Calculate melting temperatures from unfolding data obtained from fluorescence Calculate aggregation onset temperatures from static light scattering Calculate trends relating to well fto well variations Use the Advanced Analysis Package if you wish to Analyse data from samples which have different spectral characteristics i e tyrosine and tryptophan using different analysis methods Change the default analysis methods to better suit your requirements Develop new analysis methods capable of performing a wide range of analytical tasks Check the results of secondary analysis by looking
29. ae a a AR Rr ir er er i eR A PA ao oweegoeaeseeoeeweoeweoaweoeoeaeweeoeaaee aoa w Data to Analyse 3 2 2 2 2 2 2 2 2 2 2 a a 2 2 2 2 2 2 2 2 2 2 2 DataSet ao aga a 460 G G G Ge amp ae amp amp AB ea Be Bae aw w amp ow ww ww ww Ww w Ww Ww w amp Ww Ww w ww Hw Ww T Scatter266 oo 6 o o o o FG Fe Fe FG FF Fe GF Fe GF FG 6 Bo E E 4 4 0 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 DataType ao oaoaaaaaqgaoaoagaoaoagaoaoawaoaoaaaaaa Run Time 0s boob O O O O O O O O O O O Q Q Q Q Q a O0 O GOO OGOMMAD DUO O OOA OOO he Oo OO OOOOOOOOOOOOOOOOOOOOO Sample Reference Run TrainingData gt QualiticationData Analysis Method Analysis Parameters Analysis Results General Statistics Category Sample Reference Replicate Statistics Combine Runs Yes No General Statistics Results Trend Analysis Group By Duplicate Debye Plot To analyse this data e Select the Analysis Collection that you wish to analyse in this case InitialPrimaryResults is selected Saved Analysis Collections a4 TE 7 gt lt Z lt 2 A O Lu ale HANDBOOK IntialPrimaryResuts AllsecondaryResutts DuplicateDbata My AnalysisList e Select the method you wish to use typically you would use General Statistics with Duplicate Data Replicate Statistics with Replicate Data Trend Analysis with Trend Data Debye Plot with Deb
30. are acquired When the Run is finished go to the Optim Analysis software to begin analysing the data and load the run Perform Primary Analysis fo produce a set of unfolding curves and scatter data Perform Secondary Analysis to find the equilibrium midpoint of the Unfolding curves and the temperature of aggregation onset Compare these results to find the more stable formulations for the molecule of interest When finished Shutdown Optim using the software Wait until all the lights go red and the blue light goes off Then it is safe to turn off the switch at the back Use the menu option File gt Exit to close the Optim Client Software Save any exported files in a new folder Shutdown the desktop PC If pictures are more your thing overleaf is a flowchart which guides the Analyst user through this process Simply follow the arrows and make a decision anytime you reach a blue diamond Avacta Analytical Ltd 2012 Aya cta agea ANALYTICAL a lt i 2 oS a O NN 5 Z w Q TA O SZ O wal m Z lt E ac gt Z U Se O 7 INAWNALSNI ANY Jav MOOS INJO Analyst Experiments Choose Experiment Analysis Software User Runs NEW Already Edit title MCA configuration Analysed View Spectra View spectra before analysis Measurements View list of chosen measurements Option to change parameters Load samples into Optim View temper
31. diffraction This spatially separated image of the light leaving the spectrograph is focused onto the pixels of the CCD with different pixel positions corresponding to different P l Avacta noe ANALYTICAL Avacta Analytical Ltd 2012 a lt i 2 oS a O NN 5 Z w Q TA O SZ O wal m Z lt E ag gt Z U W O O 7N INAWNALSNI ANY FAV MAOS LNANO wavelengths The soectrograph has a focal length which in association with the size of the pixels in the CCD and the diffraction grating dispersion determines the pixel resolution Bear in mind that the achievable spectral resolution will also be affected by the choice of slit width A wider slit increases the light throughput at the expense of the final resolution Inside the spectrograph is a diffraction grating Light incident on this grating at a known angle angle of incidence is diffracted and reflected at an angle diffraction angle which varies depending on the wavelength of light and the density of the grating The centre wavelength is defined as the wavelength whose diffraction angle causes it to be imaged in the centre of the CCD array Regardless of the numerical value of this centre wavelength the sum of the angle of incidence and the diffraction angle is a constant This is called the inclusion angle The CCD detector and spectrograph are joined together physically within the Optim 1000 To account for any misalignment in the conn
32. each well is measured it will be highlighted in this window File Plate ag gt Z U W O O 7N Plate l Stage MCA 1 243 INAWNALSNI ANY FAV MAOS LNANO MCA 1 MCA 2 MCA 3 Hp NEW FEATURE clicking on a well will move the instrument optics such that the selected well becomes the active well A single shot will now capture the selected well eoooooococ coe od0 00080 ooocoooo00c0 0000 GOd0 A Open Door A Close Door Run Control The Run control window contains the buttons to take a single shot start a Run or abort a Run If a Run is aborted before completion the partial set of data will still be saved to the database If you wish to restart the Run you must opt to start a New Run from the Experiment menu and re enter all of the settings as required The Run settings have been recorded in the xml file which can be accessed by clicking on the blue ID of a Run in the View Spectra tab o Single Shot i Start Run tC Abort Run When you have checked or changed the measurement settings e You can verify your settings by taking a single snapshot of each well to preview the spectrum Click SINGLE SHOT in the Run Control window P Avacta age ANALYTICAL Avacta Analytical Ltd 2012 D r Alternatively select Single Shot from the Acquisition menu or use the toolbar button e The acquired spectrum will be immediately visible in the View Spectra tab but w
33. for the chosen Experiment Runs This tab shows a list of all completed runs for the chosen Experiment in the selected Project Click on a Run to view the details in the box on the right of the screen If you are beginning a new Run it will appear at the bottom of the list G Run 51 01 04 2010 Co Run 52 01 04 2010 Go Run 55 06 04 2010 G Run 56 06 04 2010 gt Run 65 08 04 2010 CMS test run Run 66 08 04 2010 gt kia Run 67 08 04 2010 han e changessome c some c ges50 me changessome change me changessome changessome chan me changessome changes e changes e lf the most recent Run is not already highlighted click on it and click 2 EDIT to edit the title and description of the Run you are about to start e Note down the Run ID highlighted in red at the top of the Run Details window This ID can be used to find the Run again quickly using the Open Run command in the File menu and will be used in the data analysis e Choose the MCA configuration the number of formulations in the Run using the drop down list The Device selection should show the default Optim version Peas It is important to select the correct MCA volume here as each behaves differently during an experiment 1 uL MCAs have an initial optimisation step to locate the cuvette centre but this isn t necessary in 9 UL MCAS Similarly each has a slightly different thermal response so are calibrated diff
34. in 0 02 MOPS M with 0 4 M GuHCl at pH Poly IgG 1 mg mL Poly IgG in 0 02 MOPS M with 0 6 M GuHCl at pH 8 Poly IgG 1 mg mL Poly IgG in 0 02 MOPS M with 0 8 M GuHCl at pH Poly IgG 1 mg mL Poly IgG in 0 02 MOPS M with 1 M GuHClatpH8 PolyigG r There are three typical analyses that can be performed using tertiary analysis Page 1 Avacta et ANALYTICAL Avacta Analytical Ltd 2012 Qz Lu Y gt lt Zz lt 2 oO O Lu aR Vat B 1O1 S AOOGAUNVH 4 EIE m Q gt Z gt i lt n m 0 Averaging of replicates to determine reproducibility either of any primary analysis result such as the light scattering intensity at any measured temperature or a secondary analysis result like a Tm rate constant or Tagg Trend analysis looks at trending data and performs a mathematical fit to describe what is happening For example o looking at how pH affects the Tm of a protein in solution we will see some examples later o fitting a thermodynamic two state model to some chemical unfolding data to obtain tree energy change of unfolding or solvent accessible surface area Debye plots to give second virial coefficients and an estimate of molecular weight Looking at the data and creating analysis collections First go to Advanced Analysis Centre tab and the Tertiary sub tab and ensure that VIEW TERTIARY DATASETS is selected At this point it is possib
35. installed the instrument either Avacta or one of our distribution partners Installing the Optim 1000 client software It is only necessary to Install the client software if you are Upgrading from a previous software version Your instrument will come supplied with the software pre installed and you will only need to perform these steps if you have not obtained a controlling PC from Avacta Analytical or if you reinstalling after a Microsoft Windows system failure P 17 Avacta ad ANALYTICAL Avacta Analytical Ltd 2012 ANONN The Optim 1 1 database is not compatible with the v1 5 software If you have upgraded your software it is not possible to load previously acquired data into the new version of the client software As a result the run numbers will reset to 1 The old version of the software is still present on the system so this can be used as before but will not communicate with the instrument Insert the memory stick into the desktop PC that is attached to the instrument a 8 m 23 gt re gt Z To install the Optim 1000 software v1 5 simply double click on the Clientinstaller msi application This will install the application onto the desktop PC that came with your instrument Chentinstaller Type Windows Installer Package The installation software will then start to install the Optim client software and if you are updating a new updated database also onto your computer The softwar
36. lt Q LU ale As such choosing a subset from the subset menu will enable the user to choose which particular trend to display When there are two results for an individual trend such as two transition temperatures these will be displayed in different colours as shown below TransTemp BarycenFluo C Conc Additive 8 Component Additive Avactad ANALYTICAL Avacta Analytical Ltd 2012 Vat B O1 S Hovering over each dataset shown will display the results for that particular transition temperature trend When displayed as a table the different trends will be labelled as Result No Trend Parameter Subset Result Wo Fit Type Fit Parameter 1 Fit Parameter 2 Fit Parameter 3 AOOGAUNVH Additive 1 Component Additive Additive 1 Component Additive Additive 2 ComponentAdditrve Additive 2 ComponentAdditive Additrve 3 ComponentAdditive Additive 3 Component Additive Additive 4 Component Additive Additive 4 Component Additive Additive 5 Component Additrve Additive 5 Component Additrve Additive 6 Component Additre Additive 6 Component Additive Additive Component Additive Additive Component Additive Additive 8 Component Additive Additive amp ComponentAdditive Additrve 9 ComponentAdditre Additrve 9 ComponentAdditrve Additive 10 ComponentAdditive Additive 10 ComponentAdditive _ no data _ _ no data _ _ no data _ _ no data _ _ no data _ _
37. no data _ _ no data _ _ no data _ _ no data _ _ no data _ _ no data _ _ no data _ _ no data _ _ no data _ _ no data _ _ no data _ _ no data _ _ no data _ _ no data _ _ no data _ 1 2 1 2 2 2 2 2 2 2 2 2 Quadratic Quadratic Quadratic Quadratic Quadratic Quadratic Quadratic Quadratic Quadratic Quadratic Quadratic Quadratic Quadratic Quadratic Quadratic Quadratic Quadratic Quadratic Quadratic Quadratic 26 615 inf 21 045 inf 39 302 inf 53 008 inf 64 04 inf 163 79 int 25 232 inf 20 839 inf 3 7958 inf 29 311 int 43 998 int 35 7 inf 18 769 inf 12 167 inf 46 566 inf 47306 inf 45 031 inf 140 62 inf 22 2 t inf 26 181 inf 51 866 inf C 60 285 inf C 44 661 inf C 49 545 inf LC 51 556 inf C 121 55 inf C 53 322 t inf C 60 617 inf C 65 462 inf C 80 649 inf C 03 093 inf C 03 534 t inf C 65 783 t inf C P4034 inf C 42 069 inf C 45 204 inf C 47 096 inf C 11 7399 inf C B2 416 t inf C 69 172 inf C 0 021947 int C 0 035592 inf C 0 015613 int C 0 010233 int C D Q00266 inf C O 0016975 inf C 0 020793 inf C 0 035587 int C O O1 41 73 inf C 0 0065334 inf C 0 008297 4 inf C 0 010338 t inf C 0 003437 inf C 0 010007 int C 0 011947 inf C 0 012887 inf C 0 00352 inf C O 0032401 inf C 0
38. spectra By using a UV laser for high sensitivity measurements and a higher power blue laser with a broader focus aggregate size differentiation can be achieved ag gt Z U W O O 7N INAWNALSNI ANY FAV MAOS LNANO Temperature Control When choosing the temperature control settings it may be useful fo consider the following information about the logic of the software The most common use of the temperature control is for a Stepped Ramp Type Experiment It is important to note first of all that the temperature will not vary continuously as implied by the word ramp but is in fact more like a staircase A Linear Ramp Type Experiment does exactly the same thing but the temperature does vary continueously at a programable ramp rate while measurements are carried out continuously The user decides the Start and Stop temperatures and the Step Size and the instrument performs a complete set of measurements on every well at each set temperature point Decreasing the step size results in a larger set of data which will of course take longer to collect You might want to bear this in mind if you have chosen a small step size and have a large number of samples on the hot plate as the samples will be held at elevated temperatures for as long as it takes to get all the measurements This may inflict thermal stress and cause unexpected changes to the properties of the protein or macromolecule under investigation as the last samples scann
39. to which all analysis methods are applied The next section describes how to create spectra collections how to create an analysis program and how to apply it to the spectra collections Looking at spectra data Now that data has been loaded the spectra can be sorted into collections First go to Advanced Analysis Centre tab and the Primary sub fab and ensure that VIEW SPECTRA Is selected om Igor Pro 6 23 OptimAnalysis Analyse Data OptimUtilities D Optim Advanced Data Analysis Engine v1 54 Development Ava cta Manager Advanced Analysis Centre Report Generator ANALYTICAL Primary Secondary Tertiary Method Developer 2500 View Spectra A 2000 Spectra a 1500 3 8 1000 500 Ra cy 0 Wore Wide A ISAS id Ee Lael _ 500 550 600 650 700 Oo Wavelength nm Select Spectra Spectra Collections Edit Table Sample_Name Sample_Conc AllSpectra a Al 913 71 628 s 0 05 Pho Ci Mw poly IgG 1 mam SampleSpectra Select Tool ee T T T vith sClatoH 326 Wool lag gT Save i Mw Mh poly IgG 1 mg ml 2 poly IgG 1 mg ml Data Folder gt run000139 D1 19 9732 C 82 895s 0 05 Phosphate Citrate M wihOMNaClatpH5 6 poly IgG 1 ma ml copy Soph Data Folder gt run000139 E1 19 9823 C 86 322s 0 05 Phosphate Citrate M with O M NaCl at pH 6 6 polylgG 1 ma ml iil Data Folder gt run000139 F1 19 9732 C 89 72s 0 05 Phosphate Citrate M withOMNaClatpH 7 6 poly lgG 1 ma ml Data
40. well by clicking gt to move to the next well and to move to the previous well o Cycle through each ordinate by clicking 3 to move to the next ordinate and to move fo the previous ordinate o Click YANALYSE at each graph with the respective method selected o Clicking will reset the view to the first well first ordinate and first abscissa e Analyse all data in an analysis collection using a selected method by clicking ANALYSE ALL It is sometimes easier as already discussed in the previous section to see transition temperatures in data that has been smoothed or differentiated Showing the smoothed and differentiated curves in this case overlays onto the normal graph and allows a clear determination of transition temperatures 354 j 08 s 352 Pg Sa FT s oF 4 s ai i i Pi a 06 350 f e i f re o F r n Ph 05 wW E MB d lt f 3 3 i o 3 pa J 3 4 m F 03o 4 5 gt 34 a z Pa 4 J ra t 02 4 P Bee 3 342 j _ 2 Me oa A 01 Pail ka wee ka ann we k ne k we bs ante Hn nee 340 a2 Wi cael seven ant ie e 00 338 0 1 70 40 s0 100 Temperaige f C Page 1 Avacta age 190 ANALYTICAL Avacta Analytical Ltd 2012 As with all graphs in the advanced analysis the Usual options to add the graph to the report right clicking on axes to scale and copying the graph to the clipboard apply The default secondary analysis methods are further details are available in t
41. wizard mode toolbar provides some quick shortcuts to tasks that you may perform on a regular basis Shutdown Optim Analyser software Go to the Advanced analysis mode Save the currently loaded analysis study Advanced mode In advanced mode the screen Is divided into tabs and sub tabs for performing different tasks There is also a toolbar to allow you to perform common tasks easily S llli The symbols in this toolbar have the same meaning as they do in the wizard mode with an additional button which returns to the wizard P Avacta Adee ANALYTICAL Avacta Analytical Ltd 2012 8 0 0 Optim Analysis 4 Optim Advanced Data Analysis Engine Beta Development Ava c ta Manager Advanced Analysis Centre Report Generator Ae f Files Analysis Log Settings Study Study Data oan In this mode data structures can be created and removed spectra can be loaded and settings changed through the Manager tab Analysis can be performed and custom mathematical methods created through the Advanced Analysis Centre and if one wishes to eschew obfuscation the Report Generator can be used to generate reports QZ Lu Y gt lt A lt 2 A O Lu Ae HANDBOOK Before you can start to analyse data you need to configure your settings In the Manager tab select the Settings sub tab to display the current settings Dd Optim Advanced Data Analysis Engine Beta De
42. 00140 P1 Data Folder run000140 A2 Data Folder run000140 B2 Data Folder run000140 C2 Data Folder run000140 D2 Data Folder run000140 E2 Data Folder run000140 F2 Data Folder run000140 G2 Data Folder run000140 H2 Data Folder run000140 12 Edit Table 0 Add Add ALL Add Ss Results to Results to Table to Copy Table 2 R R Export To change the data that is displayed click EDIT TABLE P 101 Avacta age ANALYTICAL Avacta Analytical Ltd 2012 a4 Lu Y gt lt A lt 2 A O Lu ale HANDBOOK Optim Analysis Edit Results Table Select Parameters to Display in Table Sample Data Results Sample_Ref a LJ TransTemp Scatter266 Temperature Sample_Name Tonset Scatter266 Temperature Sample_Conc TransTemp BarycenFluo Temperature Sample_pH LJ Tonset BarycenFluo Temperature Notes Replicate_No Analyse Reference Buffer_Name Buffer_Conc Additive1_Name A A ddidh A Manna TT gt Sample data that is available to display can be checked as can results of the secondary analysis Clicking APPLY will close the edit table window and apply the settings to the results table LJ Ti LJ Ti CJ d CJ LJ LJ CJ CJ m f Results for each Cuvette Scatter266 BarycenFluo Temperature Temperature Run Well Sample_Name Sample_pH Tonset TransTemp 1 run000005 A1 41 mg ml MabThera in 0 M Histidine 0 69 5 67 846 0 13001 Sa 79 769 0 4381 A O run000005 B1 1 mg m
43. 010113 inf C 0 0096337 inf C Choosing to display the data as a table puts the results of all tertiary analysis for all subsets into a single table ais m O gt Z gt P lt n m 7 As before all results can be copied to clipboard exported to file or inserted into the report Avacta Analytical Ltd 2012 Page 80 Ayacta ANALYTICAL XV ADVANCED DATA ANALYSIS MODE The advanced analysis mode of the Optim Analysis software is capable of analysing the data generated on the Optim Client in a large variety of ways both through default analytical methods that have been developed by Avacta Analytical and by user generated custom methods Data analysed in the wizard mode is compatible with the advanced mode and the two can be switched between at will Managing the software and Loading data Loading data The first step of performing data analysis is fo load the acquired data Create a new study to keep the data grouped these could have the same name and contents as the projects in the Optim Client software To do this select the Manager tab and in the Files sub tab click in the Study section and give the new study a name Then click CREATE Report Generator Create a New Study QZ TE 7 gt lt Z lt 2 A O Lu ale HANDBOOK Study Name MyNews tudy To add spectra to our study click in the Study Data section The software will list all the run
44. 0439597 170 493744333F 05 13 31 7010 7 04 07 PMI A1 31 AC IA Nk If you have loaded a Run and there are no spectra listed in the View Spectra tab click J LOAD in this window and wait a few seconds for the list to be populated e Click on any spectrum to view it as a graph or P l Avacta age ANALYTICAL Avacta Analytical Ltd 2012 ag gt Z U W O O 7N INAWNALSNI ANY FAV MAOS LNANO e Tick the Auto Show Spectra box to view the most recently acquired spectrum e Any spectrum where the detector has been saturated by an intensity that is too high will be displayed in red e Change the view settings by typing values into the min and max boxes to the left of the graph then selecting a different field e Sort the data by clicking on a column heading Hold down shift and click up to four column headings to sort by multiple factors You can export data from this tab to Microsoft Excel files All exported files will be saved in the folder C Optim Client Export The spectra displayed will have been calibrated by default using the four calibration procedures described To export a calibrated spectrum to Excel before performing any analysis e Right click on the spectrum in the list and choose Export Calibrated Spectrum A subset of spectra can also be exported by well temperature measurement or all spectra can be exported to Excel To export many spectra e Click the J EXPORT button Clicking an option in thi
45. 2 If you are adding spectra from a run into an existing study then select the study and click 7LOAD NEW RUN DATA This will open a pop up box where you can select the run you want and click LOAD P 7 Avacta ad ANALYTICAL Avacta Analytical Ltd 2012 AOOGAUNVH 4 ais m Q z gt Z gt i lt n m 0 Optim Analysis Add Run Data Add Data to Study Data Available Run Data 3 If the study is already loaded select the study where your data is stored and choose a run from the dropdown box on the right When you have selected the run you wish to analyse then click on GOTO PRIMARY ANALYSIS to go to the next stage of the analysis Performing Primary Analysis The primary analysis can be completed by clicking Y ANALYSE SPECTRA A progress bar will appear underneath and indicate the progress of the analysis By default the system will perform analysis using the default methods If you wish to use a different analysis method such as one defined and saved by an advanced user you can select this in the droodown menu Perform Analysis __ Analyse Spectra Choose a Method _ All Methods _ When the formulation information was defined at the start of the experiment in the Client software the Analyse and Reference options were set to either TRUE or FALSE for each sample well Any sample well that had the Analyse option set to FALSE will not be analysed Any samp
46. 666 inf Curvature 0 011947 inf C 42 069 inf C The default analyses carried out here are 1 Any trend analysis such as choosing any Transition temperatures aggregation temperatures vs concentration of protein additive will be fitted with a quadratic function QZ LO Y gt lt A lt 2 A O Lu ale Vath B 1O1 S C TransTemp FluoPeakPos 0 0 0 5 1 0 1 5 2 0 Conc GuHCl Component Additive 2 A primary analysis result such as peak position at a particular temperature plotted against concentration of a denaturant such as GuHCI will be fitted with a two state equilibrium unfolding yielding a free energy of unfolding AGyn an M value which is the molar free energy change and by dividing the free energy by the M value a midpoint of unfolding P 77 Avacta Sae ANALYTICAL Avacta Analytical Ltd 2012 0s nm FluoPeakPos Run Time 0 1 2 3 4 5 6 Conc GuHCl Component Additive 3 Plotting any datatype parameter against either sample reference or replicate number as defined in the formulation information that is inserted in the client software will disolay data representing the reproducibility of the measurement In this case the dot represents the average value the box represents the statistical uncertainty and the bar represents the range of the data AOOGAUNVH 3 4 ais m Q gt Z gt Lic lt n m 0 MeanVal
47. Avacta Analytical provides innovative analysis and detection solutions to the biopharmaceutical pharmaceutical and healthcare industries through leveraging its broad ranging expertise in analytical techniques and protein science Avacta Analytical s unique skill lies in making advanced analytical technology more broadly accessible through a range of market centric products and services Our primary focus is to equip pharmaceutical developers and manufacturers with tools to get their products to market more quickly at reduced cost and to optimise the critical to performance properties of these new drugs Avacta Analytical s products and services make it possible for drug developers to analyse their compounds in much greater detail at an earlier stage in the drug development pipeline than is now possible thereby providing timely identification of problems that can cause costly late stage failures or poor product performance Avacta Analytical is a wholly owned subsidiary of Avacta Group plc and occupies its own purpose built laboratories and manufacturing facility Avacta Analytical is ISO9001 registered Avacta ANALYTICAL Avacta Analytical Ltd Unit 651 Street 5 Thorp Arch Estate Wetherby LS23 7FZ Tel 44 0 844 4140452 Fax 44 0 844 4140453 Email info avacta com Web www avacta analytical com
48. E E E er eee 35 Ole ES el O10 i g ee eee ee ee ean mn nn ne eo 35 Vik STARUNGA NEW Tr Wl aprtenccte sua cansacnndaanuuadeieacscistanosecsbeanasthaseladssacanensvens 37 OBO Iena E A E 37 Es E steerer erect ste ent cate crea cae E TE E A A A E can E E E 37 Avacta ANALYTICAL Avacta Analytical Ltd 2012 PC NS T terete eee teste ease tested tuts E E E 37 SITET OSS ae EEE EEE E E 38 PPNO e E EE Ei 39 FNS EIE EEE E ct aan EE EE one ou EE E E PEET E saunas 40 BOM MUONS a E E E 40 Preparing the Micro Cuvette Array MCA ssssesssesserssesserssessesserseesserserssersereserses 45 Loading the MCAS INIO OD HI pereee terete tee ne r aE EEEIEI 46 Taking a Measurement sessssrssessrsresrrsrerrerrerrtrserstsrersesrerstrserserecseeseeseesseseeseeseeseeses 47 IX UER MANA CEMENT a A E E E EE E 53 WS OPS A EA O E E E A A A E AEE 53 X DAIAMANACEMEN Tete rece enen ian N EE AAAA AE Ei 55 COMON are EREE E A A E 55 PO E A T eauvecieaantacicdanveciaasvasiedanvecneacovaciess 56 Buffers and AdditivesS eseessessesseeseeseeseererssrseeseesseserssesersseseeserserressessessessersesrerseererss 56 XI EXPERIMENTS ETIN G re E 59 EO Si So EE E E E 59 lemperature gi O kersaan ENE ESEE E AE EN 60 CMO TOTS e E E EA E ceases 6 THE OPTIM ANALYSER HANDBOOK cccsccsccsccsccsccsccsccsccecceccsccesceccessescesees 65 Alls CCE VING SIA ME x coscnaaiaescaddnaesenacauesaasanuetensancescadanaeteaaesuesamiacueimeeartestecmocstosateosnd
49. Facies Physical Injury Hazard Do not attempt to move or lift the instrument without the assistance of others the use of appropriate moving equipment and proper lifting techniques Improper lifting can result in painful and permanent back injury Depending on the weight moving or lifting an instrument may require two or more persons Poorman If the Optim Instrument is moved after it has been installed it must be re validated by an Avacta qualified service engineer before use Moving and Lifting Stand Alone computers and Monitors A CAUTION Physical Injury Hazard Do not attempt to move or lift the computer or monitor without the assistance of others Depending upon the weight of the computer and or the monitor moving them may require two or more people e Make sure you have a secure comfortable grip on the computer or monitor when lifting e Make sure that the path from where the object is to where it is being moved is clear of obstructions e Do not lift an object and twist your torso at the same time P 12 Avacta age ANALYTICAL Avacta Analytical Ltd 2012 e Keep your spine in a good neutral position while lifting with your legs e Participants should coordinate with each other before and during lifting objects Ensure that everyone who operates the instrument has received instructions in general safety practices for laboratories and specific practices for the instrument Electrical Safety The Optim 1000 i
50. Folder gt run000139 G1 19 9823 C 93 244s 0 05 Phosphate Citrate M with 0 05 M NaCl at pH 2 E poly IgG 1 ma ml To display spectra e Inthe region at the bottom of the screen entitled Select Spectra select an individual soectrum by clicking on the text not the checkbox e More spectra can be displayed by holding the shift key and clicking another row or rows Avacta Analytical Ltd 2012 OAvac Page 87 QZ LU Y gt i lt Z lt oO O LU T HANDBOOK Edit Table R Well Temperature Set Temperature Laser Power Run Time Exposure Time Time Stamp s a B1 56 0183 C T 2 02696e 05 5850 53 s 00 00 00 00 01 01 1904 C1 56 0183 C 2 0452e 05 5853 95 s 00 00 00 00 01 01 1904 8356 C EJ i EE Sort Too PLIM E D1 19 0306 H0 L Woar D S E1 2 00826e 05 5861 29 s A ET 5964 97 s G1 i 2 0422e 05 5868 68 s 00 00 00 00 01 01 1904 H1 56 0183 C i 2 04959e 05 5872 34 s 00 00 00 00 01 01 1904 Check to add to spectra collection Select to add to graph display There are two clickable regions in the list of soectra By checking a box in the column entitled Collection the soectrum can be added to a spectra collection By clicking on the text in the row the spectrum is displayed in the graph The colour of the background in the collection column matches with the colour of the trace in the graph e EDIT TABLE clicking this will allow you to select whi
51. H To create a custom analysis collection 4 ais m O me gt Z gt P lt n m 7 e Create the graph that you wish to add to the analysis collection o Selecta dataset which is a primary analysis result dataset such as Scatter 266 o Select a datatype Tonset TransTemp data at a particular temperature o Choose samples from the list under Select Results to Display or Add to a Collection This will disolay curves in the plot display area as you have selecteq e Click ADD TO COLLECTION e Repeat until the collection contains all the information you wish e Data can be removed trom the collection by clicking il REMOVE FROM COLLECTION e Click SAVE COLLECTION As in the primary and secondary analysis sections you can use the EDIT TABLE SELECT TOOL and SORT TOOL functionality to assist in the making of graphs Performing tertiary analysis Go to Advanced Analysis Centre tab and the Tertiary sub fab and ensure that ANALYSE is selected P l Avacta ogee ANALYTICAL Avacta Analytical Ltd 2012 fi 1 h Igor Pro 6 23 ce f aa OptimAnalysis Analyse Data Optimtilities Optim Advanced Data Analysis Engine v1 54 Development Aya cta ANALYTICAL Manager Advanced Analysis Centre Report Generator Method Developer ool a ot D ed Primary Secondary View Tertiary DataSets no Gi
52. HNS WZ UNM W eyayo ayaydeoyd g0 0 T E T O C i JO HE OHNO WZ UM WN eauyo apaydeoyd s0 0 E C n O O He yw tone WZ uam W aayo ayaydeoua so o 5 TE Ey C O PUA JOE eon 21 UM N opaugo apadeoud s00 3o gt o O O S o Hamona wz uw y mao apaudeoyd so o 5E mS 2 DC GH LO ome wwionne neum y saro audoyd soo zt E D O O LP 0 Hd W IOHNS WZ L UNM W OyaIyo Baydeoud S0 0 T 2 me O 0 HA W ISHND WZ UNM W BIIYO BJUdIOUd 0 0 a E O LM OHARIN WZ uym w eayo epydeoud 0 0 eo Se ormo nrun sousoua soo 2 E Q O e HIWI NEUM W oayo opoydooyd 80 0 T S D J 0 o OHAR OHM Wz am W eaayo ayaydeoud 60 0 Z gle oO c Wis 0 HAW IOHNS W 2 UNM W ByaIyo aaydeoyd S00 Z elo t C i 0 Hd W IOHNS WZ UNM W B8IYO BUAIOUd SO O alol I O l 0 Hd W IOHNS WZ UNM W B9IYO B1UAIOUd SO O Sle 2 O z EE EET Hd 48 OHS W 2 UYA W 0aIyg oyaydeoyd wos E 5 z fe ove worn ne vu n onnados a B G A 0 Hd W IOHNS WZ UNM W yByO BywUdNOUE g0 0 SE z a g mi cot A PS O E E E y5 i 2 oyu Oo WW OUND 0 OmOLUL Wine So zeqedS THE OPTIM ANALYSER HANDBOOK Page 108 Avacta ANALYTICAL tuning of the analyses The available parameter depends on the particular method Each of the tertiary analysis methods has parameters associated with it to allow and these are detailed here Avacta Analytical Ltd 2012 aes Pownce omen onion General Category Sample Reference Changes the displa
53. IgG TRUE 1 mg mL Poly IgG in 0 02 MOPS M with 2 8 M GuHCl at pH Poly IgG TRUE 1 mg mL Poly IgG in 0 02 MOPS M with 3M GuHClatpH8 Poly IgG TRUE 1 mg mL Poly IgG in 0 02 MOPS M with 3 2 M GuHCl at pH 8 Poly IgG B2 1 mg mL Poly IgG in 0 02 MOPS M with 3 4 M GuHCl at pH Poly IgG 1 mg mL Poly IgG in 0 02 MOPS M with 3 6 M GuHCl at pH Poly IgG 1 mg mL Poly IgG in 0 02 MOPS M with 3 8 M GuHCl at pH Poly IgG 1 mg mL Poly IgG in 0 02 MOPS M with 4 M GuHClatpH8 Poly IgG 1 mg mL Poly IgG in 0 02 MOPS M with 4 2 M GuHCl at pH Poly IgG 1 mg mL Poly IgG in 0 02 MOPS M with 4 4 M GuHCl at pH Poly IgG 1 mg mL Poly IgG in 0 02 MOPS M with 4 6 M GuHCl at pH 8 Poly IgG 1 mg mL Poly IgG in 0 02 MOPS M with 4 8 M GuHCl at pH Poly IgG 1 mg mL Poly IgG in 0 02 MOPS M with 5 M GuHClatpH Poly IgG 1 mg mL Poly IgG in 0 02 MOPS M with 5 2 M GuHCl at pH Poly IgG 1 mg mL Poly IgG in 0 02 MOPS M with 5 4 M GuHCl at pH Poly IgG 1 mg mL Poly IgG in 0 02 MOPS M with 5 6 M GuHCl at pH Poly IgG 1 mg mL Poly IgG in 0 02 MOPS M with 5 8 M GuHCl at pH 8 Poly IgG 1 mg mL Poly IgG in 0 02 MOPS M with 6 M GuHClatpH8 Poly IgG Water M at pH 7 HE HUHH ELE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE TRUE HOGE TRUE TRUE Pb EE EO Ed EO Ed HH G GHH HEE 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0 eoece o5 o5 o5 og o og CKBSC eee o g eC eee eS a Et Et Et ES u EF ES bs a ka E G G aU GO
54. Lines and markers the graph will be displayed with both markers and connected by lines 00 Optim Analysis Analysis Wizard Optim 1000 Analyser Wizard Primary Analysis Avacta ANALYTICAL Select Data to Analyse Perform Analysis Choose 7 a apa Opt001 Protein Standards v Analyse Spectra Choose a Method _ All Methods _ rund00047 View Primary Analysis Results a eerie goke MAAN TOA Select X axis Temperature Exporting Export Choose Data to Export le AA Pe 28 es a Ze iai T Reporting Add to Report Temperature oC Choose Plot Data to add to Report All Ratio350_330 Select Y axis Ratio350_330 G ON mapaa parga aeaea O goe Lines oan azo s20i c2u E2 0 F2 m c2m H2m 12 m 22 Ekz m2m n2 02 P2 m asm esm csm csm F m com Hsm is mjs mkom vse Nsmosm Ps m Go to Secondary Analysis D gt The colour of the points and lines that are displayed on the graph corresponds to the key at the bottom of the graph If you wish to add or remove results from a P Avacta agai ANALYTICAL Avacta Analytical Ltd 2012 a4 UO Y gt lt A lt 2 A O Lu As HANDBOOK AOOGAUNVH ais m Q gt Z gt i lt n m 0 particular well from the graph you can click the coloured square next to the well reference in the key t
55. OptimDB Files Capture Data that is stored in studies are by default saved in a folder called Optim Studies that can be found in the users Documents folder and accessed either directly or via a shortcut on the desktop If you are installing the analysis software on a computer that is not connected to an instrument and you wish to load data from a different location please refer to later sections which provide information on how to achieve this Page 21 Avacta a ANALYTICAL Avacta Analytical Ltd 2012 Z O lt o lt fa FE a a a 8 m 23 gt a gt Z Activating the analysis software The analysis software features a copy protection dongle that requires initial activation before analysis can be saved or exported To activate the software select the OptimAnalysis menu and the Update Licence option Optim Analysis Licence Utility Licence Activation Utility Dongle Number 1974691019 Number of Activations Enter Activation Code Feature Licence Expired 0 2 4 4 5 B f E You should then contact Avacta Analytical Ltd via optim avacta com with your name Optim serial number the dongle number and the number of activations shown above Avacta Analytical will send an activation code that will enable you to use the software without limitations while the dongle remains inserted If you remove the dongle you will not be able to save or export data from the software
56. P 1 Avacta ogee ANALYTICAL Avacta Analytical Ltd 2012 ll SAFETY ADVICE For your protection and to avoid malfunction of your Optim 1000 observe the following safety precautions Read and understand all the information in the instructions for use and observe all warning and caution statements Failure to do so may result in damage to the product injury to personnel or poor instrument performance The following table describes the symbols that may appear on Optim instruments Each symbol may appear by Itself or in combination with text that explains the relevant hazard A Indicates that you should consult the manual for Z O a lt o lt fa FE a a further information and proceed with caution Indicates the presence of an electrical shock hazard and to proceed with appropriate caution Indicates the presence of a hot surface or other high temperature hazard and to proceed with appropriate caution A Indicates the presence of a laser hazard and to proceed with appropriate caution Indicates the presence of moving parts and to proceed with appropriate caution Indicates the presence of a biological hazard and to proceed with appropriate caution Do not dispose of this product as unsorted municipal waste Follow local municipal waste ordinances for proper disposal provisions to L_ reduce the environmental impact of waste electrical and electronic equipment WEEE gt gt gt e D a m
57. Project the user must choose one of the experiments to run There are four different types of experiment available The experiments contain pre programmed Page 31 Avacta age 3 ANALYTICAL Avacta Analytical Ltd 2012 a lt i 2 oS a O NN w Q Sy Ze OR SZ O O wal m Z lt T AE gt Z U W O O 7N INAWNALSNI ANY FAV MEOS LNAMO settings designed around characterising certain properties of samples for typical Optim applications The diagram below is a simplified view of the structure of the software At first glance this may appear complex but many of the processes illustrated here are default settings that the user need not be concerned with Further detail on all of the stages depicted can be found further on in the manual if you stay awake to read that far A more experienced user can use the diagram to understand how settings and data are handled by the software Square edge boxes represent processes that happen automatically whereas round edge boxes indicate that the user must interact with the software in some way In this diagram the term user includes all levels of user Please refer to the Glossary at the end of this guide for other definitions P 2 Avacta age ANALYTICAL Avacta Analytical Ltd 2012 AOOAUNVH INAWNALSNI ANY JAVMIAOS LNAND NF IS AS sanes sasn ETF EK Jl ld Sunsey pesAjeuy pijads pasAjeuy NNY YILJV ASVEVIVO Jere r ere
58. SE 40 OHO AfIsUd U UNSW OULUSDAIDg JI JOOC UDIZUDJOF AjISUS UI HUUSLJO9DS pSjoubelu ULBUS OADM SA ALISUS IU JO Hul sisuUOd DILDEdS S GWUDS OUI Ul UISLOJA SUL JO DINJONALS AJOIUO SUL IO SLUBLUOD UOILNJOS JO SSOLW JONS ow UDSW JOULISO O4 S 40 1 Joy LUNMILOEdS PSINSOSUW WOJ SIOLOWIOIOA LOOILXA AJOUUd SINAINO NDJOQ SOOULOW jINOJOq The data can be viewed in a data structure that is made up of A study 2 Arun l 3 A series of collections comprising either a Spectra P 9 OAyacta n Avacta Analytical Ltd 2012 AOOGAUNVH 4 aie m O mA gt Z gt Lins lt w m 7 b Primary analysis results c Secondary analysis results d Tertiary analysis results Data that Is stored within a collection is passed through a method and that generates output that can be added to a separate collection and analysed by a subsequent level of analysis Only the study is a rigid entity A study can contain data from many runs in the advanced analysis mode these can be analysed simultaneously A collection can contain many different members whether they are spectra unfolding curves etc and the members themselves can be in many different collections In the wizard mode the concept of collections is of little importance as the user is not able to define their content All data is passed to the analysis engine in this case For further information about the default methods the outpu
59. Spectra Run Time Spectra Exposure Time Spectra Time Stamp Sample Sa W Graph E Collection e SORT TOOL a tool fo allow sorting of soectra by any of the parameters in the table o Under Create New Rule select which parameter you wish to sort the spectra list by e g set temperature well reference etc o On the right select how you wish to sort the spectra Click ADD to add this new rule to the current rules o To add the new rule before or after the selected rule in the Current Rules click BEFORE AFTER The word in bold represents the current choice To delete a rule from the Current Rules first select it then click DELETE o To apply the sorting click APPLY The example below will sort the spectra list by ascending alohanumerical well reference all Al well soectra will be shown first then all A2 soectra etc followed by ascending alohanumerical set temperature All Al spectra will be displayed first in increasing temperature order followed by A2 spectra in ascending temperature order etc Optim Analysis Sort Tool QZ LU Y gt i lt Z lt Q LU ale Vat B O1 S Rules for sorting spectra in Table Current Rules Parameter Spectra well Spectra Set Temperature Create New Rule Sort Method ascending alphanumerical ascending alphanumerical Add Before After Delete Spectra Set Temperature Laser Power Run Time Exposure Time Avacta Analyti
60. a is the same as displayed above and as such the difference in the way the data Is presented is clear repeated below FluoPeakPos nm 20 40 60 80 100 Temperature C The advantage of displaying differentiated data is that it is sometimes clearer to the observer where the transition temperatures are due to them being at the position of a peak maximum rather than the steepest point in a curve However by displaying the data in this way important information about tertiary structure indicated by the start and end points of the undifferentiated curves Is lost P Avacta age 7 ANALYTICAL Avacta Analytical Ltd 2012 QZ LU Y gt i lt Z lt Q O LU T Vath B 1O1 S AOOANVH 4 EE m O v lt gt Z gt oa lt A m s Performing the secondary analysis Go to Advanced Analysis Centre tab and the Secondary sub tab and ensure that ANALYSE is selected hi Igor Pro 6 23 So OptimAnalysis Analyse Data OptimUtilities i Optim Advanced Data Analysis Engine v1 54 Development Ava cta Manager Advanced Analysis Centre Report Generator ANALYTICAL Primary Secondary Tertiary Method Developer View Results of Primary Anslysis E Results E g 20 lt 9 60 80 100 Temperature C Saved Analysis Collections Plot to Analyse Analysis Method Results AllPrimaryResults Run run000172 Tm_Method_153 a Characteristic Va
61. an ap aeaee 0 cuecemeeaceammnaias pHo pHo pHo Scatter266 Run Time 0 s counts nm eee eFeeeee eee e i Results CoO oO oO oO oO oO oO oOlUCUUCUCUUCUCUUCCBCCWKOUCUCOOCCcUOCUCOCoOcUCUUCOCCcUOCUCOCCcOUlCD a ar f L TIT Ir IIIZ IIZ IZIrIZIZIZIZIZIZIZIZIZIZIZRIZRIZ ZEIT T i aasnaaoeaeeaonnahaonhaaonhaoaoanhaaaoaateeteereeeeemeemee eee ee eee Se es Saved Analysis Collections SbOGGGG FFG FCG GF GTFTGFTC TCU UGKUTUCUUTUCUGGCUKGOUCUGKUCUCUGUCUCUGUCUGUUMW FT FF EZEZ EZEZ SF FSFE SFESFSF FFF FFB FZ E L ee i 2 2 a a a a ee IIZI a AllSecondaryResults Z Z Z EZ EZEZ 222222222 222222222 mH OOOO Oo ono one Oo om mm i NUN NNN NNN NNN NNN NNN NNN NNN Db g g wo wo w w O w w O O O O O O O O O O g g g DupicsteData i ili o eceecererererrererererrere rrer errrerrerre ooo oO 6 6 ob 6 6 bob 6 6 i E E E amp amp amp amp amp amp amp amp amp amp amp amp amp amp amp amp amp amp amp amp amp sone sc HTOCUCUC TOC OOlCUCU OCOCUCOCOlCUC TOC OC TOCOlCUC OC TOCOCUCUCOCOCUFVOChlU ShCU HS MyAnalysisList 88 3 3 Ss Ss FB Fs FS BEBE 5 Z amp 3 3 3 Z amp amp 3 8 5 ZSEEEBOPEPBEPBEEEBEBEBEEBEBEBEBEBEBEBEBEBEBZEBZEE 22H 2eeeeBRBRB RPP ReERBERSEESESETCESEHECSE SE oe 2s f 2 f 2 Sf 2S 2 2 2 FS 2 SF 2 TF eS SS SF eS le E E 8 8 ff ff fF FF fF FF SF EF BE EE EB Aa er aR
62. anging the samples Workstation Safety Correct ergonomic configuration of the workstation can reduce or prevent effects such as fatigue pain and strain Minimise or eliminate these effects by configuring your workstation to promote neutral or relaxed working positions Page 14 Avacta a ANALYTICAL Avacta Analytical Ltd 2012 Fc snintas Musculoskeletal and Repetitive Motion Hazard These hazards are caused by repetitive motion awkward posture holding static unhealthy positions contact pressure and other workstation environmental factors To minimize risks use equipment that comfortably supports you in neutral working positions and allows adequate accessibility to the keyboard monitor and mouse Chemical Safety Chemical Hazard Warning Z O lt o lt o FE a a Teens Chemical Hazard Before handling any chemicals refer to the Material Safety Data Sheet MSDS provided by the manufacturer and observe alll relevant precautions Pca Chemical Hazard Chemicals in the instrument including liquid in the lines are potentially hazardous Chemical manufacturers supply current Material Safety Data Sheets MSDSs These contain information on storing handling transporting and disposing of the chemicals safely To minimize the hazards of chemicals e Read and understand the Material Safety Data Sheets MSDS provided by the chemical manufacturer before you store handle or work with any ch
63. at the data superimposed with the primary data Analyse data from multiple runs simulfaneously P Avacta ogee ANALYTICAL Avacta Analytical Ltd 2012 THE OPTIM ANALYSIS PHILOSOPHY In this section the data flow around the system and the definition of the different XIII analysis levels are defined There are three levels of analysis in the software Each of the levels has three attributes inputs methods and outputs qd 9194U1 DUD SLUSIDDIS5 SLUDIDIJOOO JOUIA PUODSS NAN MAy g Nnjoa W PUD Noy Ju IdIox JO UOlLOIJUSDUOD IJsuIOHO JOIJUSISJJIP yeno q DILOJPON UOISseJOSI JOSUN SUL BINJOJOdW9a JOINDIOd O 1D JINSOJ sIsAJOUD AJOWLd sqo gt 660 l L O19 SUOILOJLUSDUOD Hd 11 OQ SUOILIOUOD jOoIsSAUC JUSIOIIIO JOMOUN SPUSIL 104 Hupjoo Jo ssodund y4 JO S AWUIOS O4 JAUWDPS WOJ SUOILOUIDA 40 OO OL DIH AOOPUNVH AASATVNV WiLdO JHL II JOILUBDUOAXS BIQNOHP JO 9 Ouls JOULIO UBNoU y SINLOJOdWd SUIOBDO JOUSIOWIO YBN L uo0l 09 9pP 6p yBnoryy SUL JO DINJOJOdWwWd9 JOULIS SUIDBDO S INS 1 sisAjOUD AJOUWWUd ou BulApjdsio Aq p sodwoo SOAIND UOILOHasIH60 DUD SSAIND Hulpjojun SISAJOUD AJOWUG SUL AQ PeloJeUsH SOAIND S1S PUD Bulpjoljun WOJ SLUDJSUOD SII JO SOINJOJIOALWS J suUoO SLUIOC Pulau LOOILXI NIOPUODES pojsl SPOUJOWW SU WOI P9 9014X9 SIOJOWIOIOM SUL 104 LUNIJDOAS ydo JO NJOA Y OES O
64. ate the median value to use as the solvent in the calculations Wells Allows user to choose an individual solvent well to use Auto Rayleigh ratio If possible will contain a list of known solvents with the Rayleigh ratio automatically adjusted for wavelength Water Uses the Rayleigh ratio of water automatically adjusted for wavelength New Selecting this allows the user to enter any value dn dc 0 185 ml g New Duplicate None Avacta Analytical Ltd 2012 Value of this parameter re lt O O 5 Allows the user to enter any value How to group duplicate data P 11 Avacta age Vie ANALYTICAL average g Mean Median Viewing the results of the tertiary analysis Ensure that the Advanced Analysis tab and the Tertiary sub tabs are selected and that RESULTS has been selected or Pro 6 23 E OptimAnalysis Analyse Data OptimUtilities Optim Advanced Data Analysis Engine v1 54 Development Manager Advanced Analysis Centre Report Generator Primary Secondary y ne Ayacta ANALYTICAL Tertiary Method Developer Tertiary Analysis Results Result No Fit Type Fit Parameter 1 Fit Parameter 2 GuHCl Component Additive _nodata_ 1 Denature 14 442 0 66943 kJ mo 7558 6 296 58 J mo Fit Parameter 3 Run Well s run000172 A1 B1 C1 D1 E1 F1 G1 H1 11 J1 K1 L1 M1 N1 01 P1 A2 B2 C2 D2 E2 F2 G
65. ature ramp settings Analyse aren Spectra Primary Secondary Results results Check settings Export to Excel View Spectra View spectra before analysis Results View list of analysis results Generate Change Yes settings PERE R RRR RRR RRR REPRE RRR REPRE RRR REE RRR RRR RRR RRR REPRE REE REE E Button command Optional stage tC PPR PPR Pee eRe eee eee eee eee eee eee eee eee eee eee eee P Avacta aeae ANALYTICAL Avacta Analytical Ltd 2012 Vill STARTING A NEW EXPERIMENT This part of the manual covers the basics of operating the instrument It includes alll details that are relevant to an Analyst level user to step them through the process of loading samples into the Optim 1000 instrument making a set of measurements analysing the results and producing a report The more challenging task of interpreting the results will be left to a more knowledgeable scientist tyoe person and will not be discussed in this manual Before an Analyst can start a new experiment the Administrator must set up the new user s login details and create a Project in the database To Begin Ensure that Optim is switched on e You should see a ring of blue LEDs and a row of green lights on the front of the instrument one may remain amber until the software is started If some or all of the lights are off please refer to Section for Instructions on how to power up the instru
66. cal Ltd 2012 ascending alphabetical descending alphabetical descending alphanumerical Avacta ANALYTICAL Page 89 AOOGAUNVH 4 ais m O F lt gt Z gt P lt n m 0 Once you have chosen and checked the spectra to analyse click SAVE COLLECTION This will allow you to create a spectra collection with a name of your choice It will then be displayed in the Spectra Collections list Spectra Collections AllSpectra Samples pectra Butters pectra SampleB utters pectra Sample oburfers pectra You can rename a collection by highlighting it in the list and clicking 4 or delete one by clicking T The will generate the default soectra collections The default collections are AllSpectra contains all soectra BufferSpectra contains all soectra with REFERENCE set to TRUE SampleSpectra contains all soectra with REFERENCE set to FALSE and ANALYSE set to TRUE SampleBufferSpectra contains all soectra in SampleSpectra for which the software can find a buffer soectrum to subtract SampleNoBufferSpectra contains all soectra in SampleSpectra for which the software cannot find a buffer soectrum to subtract Clicking on COPY GRAPH DATA will copy the data in the graph to the clipboard such that it can be pasted into other software Clicking on El will add the graph on the screen to the currently open report This is discussed in more detail later in this handbook
67. cap with the clip AN Nwarnine Biohazard Chemical Hazard Your sample formulations may contain components that have potential to transmit infectious diseases or are dangerous chemicals Follow all appropriate local and national regulations Wear appropriate protective eyewear gloves and clothing when handling such samples Loading the MCAs into Optim e Open the Measurements tab In the Stage Control window click OPEN DOOR to access the sample drawer This will open the door and P 4 Avacta age Ae ANALYTICAL Avacta Analytical Ltd 2012 move the stage into a position where it is easily accessible from the door Hp Alternatively click the OPEN button on the Toolbar g 1 Wait for the door to open fully and then pull the drawer out to its full extent The LED will flash intermittently red and green 2 Place the MCA sample holders onto the hot plate sequentially from left to right ensuring that each is secured by the brass spigots and that each is flat with no gap between the MCA base and the copper 3 Place the insulating lid over the MCAs 4 Push the drawer all the way back in and check that the door LED flashes green a lt i 2 oS a o O NN w Q mA OR SZ O O wal QO Z lt IE e Inthe Stage Control window click CLOSE DOOR to close and lock the door and return the stage to its default position HH Alternatively click the CLOSE button on the Toolbar A
68. ch parameters relating to the samples are displayed in the table such as the well reference the temperature the sample name pH etc Simply check the field you wish to display then click APPLY Optim Analysis Parameters AOOGAUNVH Display Parameters Parameter Names ais m Q F gt Z gt i lt n m 0 M4 Set Temperature Ml Laser Power Run Time BA Exposure Time Time Stamp L Sample_Ref Ml Sample_Name IL Sample_Conc Ml Sample_pH IC Notes 3 LJ Replicate No e SELECT TOOL this tool will enable you to select spectra to display or to prepare to add to a spectra collection for analysis o Choose to group the spectra by the well reference temperature sample name pH etc then select the value you wish in the right hand list o Choose whether to add the selected spectra to the disolayed graph check Graph a spectra collection check Collection or both o Select whether to add or remove the selection to or from your displayed graph or spectra collection by selecting ADD or REMOVE respectively To start a new graph or spectra collection click NEW In the case of the example below all soectra with a set temperature of 18 C will be selected Avacta agece ANALYTICAL Avacta Analytical Ltd 2012 Optim Analysis Select Tool Select Spectra Choose Parameter amp Values Parameter Run Spectra Well Spectra Temperature Spectra Set Temperature Spectra Laser Power
69. ct Set o To reset to the calculated value select Reset o Toremove the result altogether if a false positive select Remove e f you wish to add a result at a particular point hover over it with the pointer right click and select Add Result From the following menu select the type of result you wish to add In order to add a result of a particular type you need to analyse the saved analysis collection you are looking at with the relevant method first e Toremove all of the analysis results click T e You can remove results in the table by right clicking and selecting Remove e You can change the order of the results potentially important for tertiary analysis by clicking the button 123 normally the transitions are ordered on significance P 99 Avacta Sa ANALYTICAL Avacta Analytical Ltd 2012 AOOGAUNVH ae m O mv a gt Z gt ins lt w m 7 The default method will analyse the contents of the analysis collection by well ordinate and abscissa The section entitled Plot to Analyse displays the data that will be analysed In the case of the example presented above well D1 is presented from run000005 with an ordinate of barycentric mean fluorescence and an abscissa of temperature Plot to Analyse Run mundgooo0s Well C 1 Plot Scatter266 vs Temperature feo 3 To analyse the rest of the data you can e Continue to analyse them individually o Cycle through each
70. curves in the plot display area as you have selected e Click ADD TO COLLECTION e Repeat until the collection contains all the information you wish e Data can be removed from the analysis collection by clicking MREMOVE FROM COLLECTION e Click SAVE COLLECTION As in the primary analysis section you can use the EDIT TABLE SELECT TOOL and SORT TOOL functionality to assist in the making of graphs It is sometimes useful to Compare complementary datasets on a single plot You can select up to two ordinates shift click for selecting multiple ordinates and an infinite number of sample curves When you do so the values for the second ordinate will be displayed on the right hand axis The colour of traces for each well will be the same so they can be distinguished by the symbol of their marker the left hand axis is ae in this case representing the barycentric mean of fluorescence and the right hand axis is a A in this case the static light scattered intensity at 266 nm 347 346 200 345 344 150 343 100 BarycenFluo nm WU S JUN0D Q9ZJae9S w 342 30 40 50 60 70 80 90 Temperature C In an example presented here the analysis collection is created to contain the data from run000005 of the barycentric mean fluorescence and the scattered light intensity at 266 against temperature This is then saved as an analysis collection called MyAnalysisCollection P 4 Avacta age ANALYTICAL Avacta Analytical
71. d assign projects to the user a lt i 2 oS a O NN w Q Sy Ze OR SZ O O wal m Z lt IE New Users To create a user e Inthe Users tab you will see a list of the users in the left hand window At the bottom of the window click NEW e You will be prompted to enter a username for the new user e Enter the new user s personal details in the User window e Inthe Login box at the bottom you can edit the username and create a password for the new user these are not case sensitive e Click E SAVE to save all the changes on this page Assigning Projects to Users When assigning a Project to a user you must decide the user s level of access for each Project It is advisable to have just one Supervisor for each Project and to assign all other users to the default Analyst access level A Project Supervisor will be able to edit the details of the selected Project and grant access to other users but can also create edit and delete their own projects An Analyst level user can merely work on the Project and view its details To assign a Project to a User e In the Projects window of the Users tab highlight a Project in the right hand list and click ADD The Project will appear in the Allocated Projects list on the left of this window e The default access level for a user is Analyst Define the user s access level by selecting the Project and clicking EDIT in the access level box P Avacta aoe
72. dinates Scatter266 Scatter473 Fluolntensity Ratio350_330 BarycenFluo uoPeakPos 60 100 FluoPeakHeight Tempersture C Select Results to Display or Add to a Collection Collection of Selected Analyses Saved Analysis Collections Edit Table Run Wel Sample_Ref a AllPrimaryResults iM runO00172 H2 0 02 MOPS M with 4 6 M GuHCl at pH Poly IgG All_vsT emperature IM run000172 12 0 02 MOPS M with 4 8 M GuHCl at pH Poly IgG i Scatter_vsT emperature i run000172 J2 0 02 MOPS M with 5M GuHCl atpH8 Poly IgG Fluo_vsTemperature BA un00017z K2 0 02 MOPS M with 5 2 M GuHCl at pH Poly IgG ll_vsRunT ime L run000172 L2 0 02 MOPS M with 5 4 M GuHCl at pH Poly IgG m Scatter_vsRunTime A run000172 M2 0 02 MOPS M with 5 6 M GuHCl at pH Poly IgG X Looking at the curves and creating analysis collections First go to Advanced Analysis Centre tab and the Secondary sub tab and ensure that VIEW RESULTS OF PRIMARY ANALYSIS is selected Analysis collections are created by displaying the data that you wish to analyse As with the primary analysis it is possible to create a default list to analyse by clicking in the Saved Analysis Collections this will create a collection consisting of all the primary analysis results curves created against all the available secondary analysis abscissas temperature and run time QZ LU Y gt i lt Z lt A O LU T Vath B 1O1 S The de
73. dison Wesley 2001 P 2 Avacta noe ANALYTICAL Avacta Analytical Ltd 2012 2 Background Subtraction The CCD camera inside the Optim 1000 imposes a dark background signal of 600 counts onto each acquired spectrum This background level is subtracted from the raw data in the following manner o At the start of an experimental run the Optim 1000 captures a dark image with no spectral information o The signal is measured at two points on the spectrum and a straight line is fitted between these points to obtain an average background measurement o This is then subtracted from every acquired spectrum 3 Device Response Calibration The Optim 1000 provides the user with a true representation of the fluorescence resoonse of a protein sample In order to do this it is calibrated to ensure that this wavelength dependence on the digitised signal from the CCD camera does not affect the quality of soectra using a device response function The pixels within the CCD in the detector contain electrical elements which generate an electric current when illuminated with light The efficiency of this process is wavelength dependent so if a broad spectrum of light with each wavelength having the same intensity was incident on the CCD the resulting digitised soectrum would not be a flat line but would display a variation in the number of counts measured A device response calibration function is obtained by using a calibrated light sourc
74. e Results Choose DataSet tter266 FluoPeakPos Temperature 50 C nm padtenebescetsssues veseossesecssescestess ssusssessossessusseusbecssssscssinesn a J1 K1 01 P1 A2 B2 C2 D2 E2 F2 G2 H2 12 J2 K2 L22 M2 N2 02 C A1 B1 C1 D1 L1 M1 N1 1 eee rn 5 Well TRun run0001721 Clicking on will create the default collections which will vary depending on the type of data that you have At this point the Optim Analysis software will interrogate the sample information that was supplied as part of the experiment If you haven t supplied complete information regarding the composition of the samples that you are measuring you can use the sample information editor that is accessible via clicking View and Edit Sample Data in the Optim Utilities menu The software will look for patterns in this information and group data accordingly For example if you have two types of samples IgG in buffer 1 at pH 2 7 IgG in buffer 2 at pH 2 7 then there will be two different sets of data to analyse for trends in primary slice data and secondary analysis results with pH The default collections that are formed are e Initial Primary results this is a collection that contains the primary slice data that is acquired at the start of the experiment e All Secondary Results contains all datasets that have secondary analysis results such as TransTemp etc e Replicates all datasets secondary analys
75. e If is shone into the instrument and the raw data acquired is compared to the known spectrum of the light source The resulting function is stored on the Optim 1000 database and is automatically applied to all acquired spectra 4 Laser Power calibration The UV laser used within the Optim 1000 is a stable light source However towards the end of a laser s life the power output can begin to fluctuate As such a final calibration step is present in the software The laser power at the start of an experimental run is recorded and each subsequently acquired soectrum Is normalised such that the laser power at the time of the measurement Is the same as that at the start of the Run P Avacta AJEG ANALYTICAL Avacta Analytical Ltd 2012 a lt i 2 oS a O NN 5 Z w Q TA O SZ O wal m Z lt E THE OPTIM ANALYSER HANDBOOK The Optim Analyser v1 5 is a software package designed and built to automatically and robustly analyse data generated on an Optim 1000 instrument using the client software If can be installed on any Windows based personal computer and for details of doing so please refer to the relevant section of this manual XII GETTING STARTED The Optim Analysis v1 5 software features two modes of operation e Wizard mode this mode allows fast and automated analysis of the data using a simple wizard based system This is the default view e Advanced user mode allows the user to analyse and di
76. e Candidate ABC List of available protein candidates to plot ron Yes Grouping spans different runs RUNS O Grouping in different runs are treated as different atch List ABC XYZ M Wavelength Auto Z List of differentiator sample parameters For example if have trends in pH with two different concentrations of NaCI Determines wavelength of incident laser based on the available name of the primary analysis result New Selecting this allows the user to enter any value If possible will contain a list of known solvents with the refractive index automatically adjusted for wavelength Solvent R I Auto Water Uses the refractive index of water automatically adjusted for wavelength New Selecting this allows the user to enter any value Solvent Well s If multiple wells containing the solvent will calculate the mean value to use as the solvent in the calculations AOOGAUNVH Median If multiple wells containing the solvent will calculate the median value to use as the solvent in the calculations Wells Allows user to choose an individual solvent well to use WXY A list of available reference materials for example water toluene Reference Wells s ais m O gt Z gt P lt n m 7 Mean If multiple wells containing the solvent will calculate the mean value to use as the solvent in the calculations Median If multiple wells containing the solvent will calcul
77. e Name of Results File This function determines the mean x value within the given range This means each x value is weighted by the corresponding y values and the mean of these determined PeakFit InoutData Spectrum to be acted upon RangeCentre Centre of Fit range RangeWiadth Width of Fit range FitMethod Fit Method Gaussian Lorentzian Quadratic LogNormal Iterations Number of Iterations move centre PeakPositionName Name of Results File PeakHeightName Name of Results File This function fits the data within the given range to the given curve peak shape The parameters corresponding to peak position and peak height are output ExpDecayFit InputData Spectrum to be acted upon RangeMin Minimum value of Fit range RangeMax Maximum of Fit range DecayConstantName Name of Results File This function fits the data within the given range to an offset exponential decay The decay constant 1 e point is output SimpleTm InoutData Spectrum to be acted upon RangeMin Minimum value of range RangeMax Maximum value of range SmoothWidthDataPoints for Median Smooth TmName Name of Results File This function calculates the differential of the InoutData after having performed a simple box smooth and then determines the maximum of this to estimate the Tm FindPeakTm InoutData Spectrum to be acted upon RangeMin Minimum value of range RangeMax Maximum value of range SmoothWidthDataPoints for Median Smooth MinLevel Mini
78. e listed Highlight an additive in the list and use the buttons below to EDIT or DELETE it as required Administrators can add to the list of additives via the Samples tab For instructions see section X on page 55 e When you have entered all the required information click G SAVE e f you wish to use this list of formulations again there is an option to EXPORT the data to an Excel file located in C Optim Client Export To import formulations you have previously edited and exported e Note the filename that you used to EXPORT the data to an Excel file located in C Optim Client Export e Change the path in the spread sheet directory to reflect where you exported the formulation too before A Formulation Settings Spreadsheet directory C Optim Client Export Spreadsheet filename formulation xisx Is the spreadsheet data well formed F Import 4 Export e Click IMPORT To import formulations from Microsoft Excel e Open the shortcut Edit Formulation on the desktop to open the Excel tool e f you see a Security Warning at the top of the window in Microsoft Excel click Enable Content before continuing Avacta Analytical Ltd 2012 OAyacta Page 42 ANALYTICAL foe DD a a T T A PEE e You will be presented with an Excel sheet called Project samples Change the number of samples from 3 to match the number of samples you have on the Samples tab in the client software Type the numb
79. e main Optim 1000 unit needs at least 15 cm 6 inches clearance on all sides for ventilation For installation and on site repair and maintenance the Engineer will require at least 60 cm 24 inches clearance either side of the instrument to gain access to the side panels Cleaning your Optim Should you wish to clean your instrument after installation you must first follow the shutdown procedure and switch off the unit at the mains Wipe the exterior with a soft dry cloth or if necessary a damp cloth with mild detergent Environmental Requirements The site should be maintained under the following conditions The user should be aware that fluctuations in environmental conditions can cause unexpected changes in the instrument s performance For best results ensure that the temperature and humidity are maintained at a constant level Temperature 15 to 30 C 50 to 95 F Humidity 20 to 80 relative humidity non condensing This apparatus is for indoor use only Under normal operation it has ingress protection rating IP20 The instrument should not be placed in direct sunlight Electrical Requirements The main Optim 1000 unit requires connection to one standard mains socket Avacta eee ANALYTICAL Avacta Analytical Ltd 2012 The Desktop PC requires connection to one mains socket The monitor may require connection to one mains socket or it may plug into the desktop PC The Desktop PC will have its own standard powe
80. e provided in the appendix of this document P 2 Avacta Se ae ANALYTICAL Avacta Analytical Ltd 2012 a lt i 2 oS a O NN w Q Sy Ze OR SZ O O wal m Z lt T V POWERING OPTIM ON AND OFF Turning On Optim Optim 1000 has two power switches 1 Firstly at the top of the rear panel where the power and data leads enter Optim a rocker switch controls the overall power to the instrument Switching this on will activate the power to the internal sub modules including the laser sources FT ere Once this switch is set to On 1 all safety precautions should be observed 2 The second power switch is located on the front of the instrument above the service status LEDs Press this button once fo start the Optim 1000 onboard computer The UV laser reaches optimal stability after one hour of warm up time Turning on the PC The accompanying desktop personal computer is controlled independently of the Optim 1000 instrument The computer can be used without the Instrument being on but not vice versa Please refer to the PC manufacturer s manual for instructions on how to turn on the PC Automatic System Checks When the Optim 1000 has been turned on the service status LEDs towards the top right of the front panel will indicate the status of the constituent sub modules When Optim has been completely switched on and has booted up six of the indicator LEDs on the front panel should b
81. e representing the contributions to the colour from RED GREEN and BLUE Each value can vary from 0 to 65535 Changing one of the values will change the colour respectively When you have changed any settings in order to make sure that you don t have to change them again once you restart the software be sure to click the SAVE SETTINGS button in the GENERAL settings Avacta agea ANALYTICAL Avacta Analytical Ltd 2012 QZ LU Y gt i lt Z lt oO O LU T HANDBOOK AOOAUNVH 4 ae m O a gt Z gt oes lt w m 7 Colour Table 65535 S 0 In LOAD DATA settings the paths to the file locations can be configured as mentioned in Advanced mode starting on page 66 Default File Locations Studies JAVACTASERVER lt edirectedFolders daniellund My Documents Optim Studies Run Data C Optim ClientDatabase OptimDe Files Capture Default Metho C Users daniel lund AppData Roaming WaveMetrics lgor Pro 6 Packages Preferences C Users daniellund AppData Roaming Waveletrics lgor Pro 6 Packages Reset It is also possible to change the location where the default analysis methods and these preferences are saved As discussed on page 52 the spectra acquired by the Optim Client software contain headers with information about the properties of the instrument at the time the individual soectra were acquired The information relates to the well from which the spectr
82. e target instrument that you wish to update P 2 Avacta oe ANALYTICAL Avacta Analytical Ltd 2012 Optim 1 5 Update n Optim 1 5 Embedded PC Update Optim Name AVAOPT O001 i It is essential that the device installer is executed on the supplied desktop PC and that the Optim user is logged into the system at the time the installer is run otherwise installation will fail due to insufficient permissions Installing the Optim Analysis software Avacta Analytical has developed a new analysis tool incorporating foundation technology called Igor Pro v 6 2 from Wavemertrics Inc To install the Optim Analysis software v 1 5 simply extract the contents of the Optim Analysis zip from the memory stick and double click on AnalysisInstallerFull msi application AnalysisInstallerFull a i Application 48 5 KB This will install the subroutines preferences files and the Igor Pro package onto your computer In order to use this software you must have a dongle for each computer on which you wish to use the software You may install the software on one computer only and any subsequent installations will require you to purchase an additional licence and dongle from Avacta Analytical The Optim Analysis software loads and saves data from defined folders on the installed computer The default locations are configured such that raw data in the form of spectra saved by the client software is loaded from C Optim Client Database
83. e will prompt you for a location to install the software Select the default location or choose a location of your own by clicking Browse before clicking Next gt 15 Optim Client 1 5 Select Installation Folder The installer will metall Optim Client 1 5 to the following folder To install in this folder click Next To install to a different folder enter it below or click Browee Folder C Program Files x86 Avacta O ptim Optim Client_1_5 Once the software has been installed you will be prompted to configure the Optim Client 1 5 To do this click Next gt in the window below Avacta Analytical Ltd 2012 OAyacta Page 18 Optim Client 1 5 Configuration Configunng Optim Client 1 5 You will then need to enter the hostname of the instrument that you are connecting to If you are installing the software on a PC supplied with an Optim 1000 instrument by Avacta Analytical then this should be automatically populated with a valid hostname Otherwise you will need to enter this here The hostname you require will be AVAOPT xxxx where xxxx are the last four digits of your instrument serial number Optim Client 1 5 Configuration Configunng Optim Client 1 5 Enter Optim name e g AVAOF T 0010 AVAOPT 0001 Avacta ANALYTICAL Avacta Analytical Ltd 2012 Z 2 lt o lt a FE a a m 8 m U gt re gt Z The installation software wil
84. ecome green The central LED should P 27 Avacta ad ANALYTICAL Avacta Analytical Ltd 2012 CLIENT SOFTWARE AND INSTRUMENT Val 1D 1O1 S ac gt Z U W O O 7N INAWNALSNI ANY FAV MEOS LNAMO change to amber IF ANY OF THE LEDS REMAIN RED CONTACT YOUR SUPPORT AGENT Turning Optim Off Foca It is advisable to switch off both the Optim 1000 and the accompanying PC when not in use 1 Shut down the onboard computer by selecting shutdown from the file menu File Acquisition Analyse Ocooog ok The blue light on the power switch upper right of the front panel of the Optim 1000 should turn off after a few minutes 2 Turn off the main power using the rocker switch near the back left of the instrument The external PC may be used after Optim has been turned off 3 When shutting down the software always use File Exit P 2 Avacta oe ANALYTICAL Avacta Analytical Ltd 2012 VI INTRODUCTION TO THE OPTIM CLIENT SOFTWARE Navigating the Software i Always switch the Optim 1000 fully on before starting the software The Optim 1000 client software controls the system during a measurement and then processes the measurement data through a series of calibration functions A typical screen is shown below Menu bar were a 9 B B lt W Toolbar Login Propects ipenments Sampie Runs Formmdstioea Mesnwements Settings View Specta Anaiyya R
85. econds between measurements and Repeat Count number e Click amp amp APPLY to apply the changes Linear Ramp For a thermal unfolding using a linear heating ramp profile type Experiment the temperature increases from a Start Temp to a Stop Temp at a constant rate that is defined by the user by Rafe All the samples are heated in the same way and at each point in time are at the same temperature Measurements are taken at each well sequentially Affer each spectrum Is acquired before moving to the next well the system waits for a period of time given by Well Hold Time This Page 4 Avacta age ANALYTICAL Avacta Analytical Ltd 2012 a lt i 2 oS a O NN w Q Sy Ze OR SZ O O wal m Z lt IE value can have a minimum value of 15 seconds as any shorter value can lead to a break down in the linearity of the heating rate To alter the Linear Ramp settings e Inthe Linear Ramp tab click Q EDIT e Type in the required Start Temperature and Stop Temperature between 15 C and 100 C e Type in the required Well Hold Time seconds between measurements of adjacent wells and Rate e Click EJ APPLY to apply the changes Stage Control The Stage Control window displays the MCA configuration that has been chosen for the Experiment which should be the same as the MCAs you have loaded onto the stage During a Run the stage moves the focus from well to well and measurements are taken at each one As
86. ection an offset angle the detector angle is determined As such in order to know which wavelength is being represented by each pixel it is necessary to know o The wavelength centred on the CCD decente o The inclusion angle between the photons incident on the grating and the diffracted photons which will reach the centre of the CCD 0 o The detector angle to compensate for mechanical tolerances 04 o The focal length f of the soectrograph and the pixel size x on the CCD o The number of lines per millimetre on the grating d These are related in the following manner to obtain the wavelength A at a numbered pixel p from the centre of the CCD A dsiwmarcsi __Aeentre 8 2d cos 2 2 A 0 0 d sin ar csi h arcta EE keen 2dcos 2 2 f pxsind Initially only the pixel size and number of lines per millimetre on the grating are known By using a calibrated light source with a known wavelength output a spectral line source it is possible to calibrate the soectrograph such that the wavelength which is centred on the detector is known Having measured this it is possible to measure the pixel at which a known wavelength occurs and iteratively fit this information to the formula above to obtain all the parameters needed to correctly scale the x axis Further information about the grating equation used to obtain this information and the optics of a soectrograph can be found in Hecht Optics published by Ad
87. ed will have been held at temperature for longer than the first The temperature that is disolayed in the Current Temp box represents the temperature inside the micro cuvette The actual temperature of the hot plate is measured directly and a calibration function is applied to find the temperature of P Avacta agen ANALYTICAL Avacta Analytical Ltd 2012 the sample in the cuvette This is accurate to within 0 5 degrees over the full temperature range When a temperature step is made the Peltier device heats the plate until the sample reaches the new set temperature within the range of the factory set tolerance which is typically 0 2 C As soon as the temperature is within this range a timer is triggered The instrument waits for the designated Hold Time before beginning the first exposure It is recommended that the user set a hold time of at least 30 seconds as this allows the samples to reach the set temperature In a linear ramp the user can set a plate hold time and a well hold time The well hold time is an option for you to add a delay between moving onto and measuring the next well The plate hold time is a delay between measuring the last well and starting to measure the first well again So the temperature will increase at a rate and the instrument will constantly move between the wells acquiring data as fast as it can If you want to you can make it pause after either each well or each complete measurement of the 3 MCAs
88. editor you will see that the input data for this function is data in dataset DO Click on the value in the editor and change it to D1 To insert this function back into the method click INSERT FUNCTION INTO METHOD A ronan ORDER IS IMPORTANT IN THE METHOD THE FUNCTION WILL BE ADDED TO THE METHOD AFTER THE FUNCTION THAT IS CURRENTLY SELECTED IN THE METHOD GENERATOR ENSURE THAT YOU DO NOT INSERT THIS FUNCTION INTO THE METHOD BEFORE DATASET D1 IS CREATED BY THE BACKGROUND SUBTRACTION FUNCTION Repeat this then for each function in the method so that they all act on the background subtracted spectra Save the method by clicking STORE METHOD and calling the method IntrinsicFluBackground To change the results that are presented to the user from the analysis method select End in the method generator and click EDIT FUNCTION PARAMETERS You will see the Function editor changes to list all of the available outputs with a checkbox Those method outputs that are not checked are hidden from the user To enable testing of the method and the display of the background subtracted spectrum check the box next to D1 and click UPDATE OUTPUT Testing a method The method that is being edited can be tested against the data that is displayed in the Preview area To test the currently loaded analysis method you should navigate to the Advanced Analysis Centre tab and the Method Developer sub tab and ensure that TEST is selected Ie oo Optim Analysis S
89. emicals or hazardous materials e Minimise contact with chemicals Wear appropriate personal protective equipment when handling chemicals for example safety glasses gloves protective clothing e Minimise the inhalation of chemicals Do not leave chemical containers open Use only with adequate ventilation e Check regularly for chemical leaks or spills If a leak or spill occurs follow the manufacturer s cleanup procedures e Comply with all local or national laws and regulations related to chemical storage handling and disposal Biological Safety General Biohazard enn Biohazard Your samples may contain components that have potential to transmit infectious diseases Follow all appropriate local and national regulations Wear appropriate protective eyewear gloves and clothing when handling such samples P l Avacta Eek ANALYTICAL Avacta Analytical Ltd 2012 e The user must take responsibility for handling their samples in an appropriate and safe manner The samples are contained with the MCA sample holders that are loaded into Optim The samples should remain contained within these sample holders throughout the course of the experiments e Once removed from Optim the samples and sample holders should be disoosed of in an appropriate manner 8 m U gt re gt Z Disposal Do not dispose of this product as unsorted municipal waste Follow local municipal waste ordinances for proper dis
90. enate Report 2 VY O v O Tabs Please select a project from the kst below Please enter the detads foe the pr t below a 5 2A TE mi O lt x I Main Window Status bar The menu bar gives access to the open close Run logout and shutdown features as well as providing a quick look list of the acquisition and analysis functions This manual can be found in PDF format via a link in the Help menu The toolbar contains a selection of the most used buttons to allow the user to quickly set up a measurement collect data and process the results These are summarised in the table below Shutdown Optim Log the user out of the Optim client program B Open the door to load samples into Optim Close the door and return the stage to its start position Acquire a single shot this will not be saved to the database Avacta Analytical Ltd 2012 OAy Page 29 Start a Run acquired spectra are saved to the database Abort the Run you will lose the current settings The B ip symbol in this guide indicates that a toolbar button or menu option can be used as a shortcut The tabs are arranged in roughly chronological order so that a user works from left to right when setting up a new experiment taking a measurement viewing the data and performing analysis Hover over a tab heading for a description of the tab contents Some buttons and features also have handy tooltip pop ups to give you a snippet of useful information ag
91. er of samples in your run and then enter then in the list which follows In order for the formulations to be correctly imported into the Optim 1000 client software it is imperitive that these samples are entered accurately Number of samples in experiment Sample number Sample name Poly IgG NATA NAYA e Add any new samples to the Sample tab e Note the example contained here has a deliberate mistake in the miss naming of Rituximab in Excel template formulation editor If such an error occurs in practice the user will be presented with an Error no sample record named Rituzimab found for well no 1 warning Please note the Sample Name is case sensitive and must exactly match the name in the database a lt i 2 oS a O NN w Q Sy Ze OR SZ O O wal m Z lt IE aa Polyc e IgG in buffered aqueous solution NATA N Acetyl L trypto phanamide Number of samples in experiment NAYA N Acetyl L tyrosinamide Sample number Sample name i me Ritisvinals Poly IgG TE Monoclonal antibody MabTher 2 NATA NAYA Rituzimab Trastuzumab yas Peres antibody herceptin Trastuzumab e Proceed onto the Create Formulation sheet e Choose the correct MCA configuration and the Number of additives from the dropdown boxes at the top of the sheet the sheet should expand or contract to show the relevant cells for editing Note due to limitations imposed by Microsoft whe
92. erently e Click GJ SAVE When you have filled in the Run details go to the Formulations tab to fill in the data about the contents of the micro cuvettes Formulations This tab is designed to store all the information about the contents of the micro cuvettes in the current Run Data can either be input directly to the database or imported trom Microsoft Excel The Excel file must be in the correct format to be compatible with the Optim software P 4 Avacta alle ANALYTICAL Avacta Analytical Ltd 2012 You should see an empty table with the same number of rows as there are wells in the MCA configuration you have chosen a Optim Vr 1 5 2 File Acquisition Help GO O BO Login Projects Experiments Samples Runs Formulations Measurements View Spectra Replicate No Sample Name Sample Conc Notes Buffer Details Additives Additive Conc M Gi Delete A Formulation Settings Spreadsheet directory C Optim Client Import Spreadsheet filename formulation xlsx Is the spreadsheet data well formed Import ne C Optim Demo E Isothermal Unfolding and Aggregation Study 3 Run 5 18 01 2012 E 16 Wells 1uL LHS To input formulation data to the database manually e Inthe Formulations box highlight the well you wish to edit and click NEW If you wish to change data that has already been entered click amp
93. eristic If empty then this parameter is ignored CharValue Value Characteristic If empty then the value is taken from InoutData This function returns OutputData whereby this data is one of the datasets in the given lists collections of datasets and whose characteristics match those supplied The Abscissa Characteristic if supplied is not required to be an exact match but instead the closest match Avacta Analytical Ltd 2012 OAyacta Page 127 ANALYTICAL QZ LU Y gt i lt Z lt Q LU ale Vath B 1O1 S APPENDICES XVII RISK ASSESSMENTS General usage 1 Sample Loading Door Ascaumon The user may trap their fingers hand in the door opening Who might be User Sliding door may trap or crush hand or fingers harmed and how What is being The door is ooened closed automatically The risk of this done already happening with sufficient force to cause harm is very low Correct operation of the door is to not leave hands fingers inside the opening when the door is closing With correct operation the risk is very low What further The user needs to be trained in the appropriate operating action is procedures necessary By whom Whene Avacta Analytical Ltd 2012 OAyacta Page 129 2 Sample stage motion A caution The user may trap crush their fingers nand with the sample X Y stage Who might be harmed and how What is being done already
94. fault collections in this case are AllSecondaryData contains all the primary analysis results sorted against all abscissas All_vsTime contains all the primary analysis results sorted against run time time zero is the start of the experiment catter_vsTime contains the Scatter266 and Scatter473 and any buffer subtracted results sorted against run time Fluo_vsTime contains the Scatter266 and Scatter473 and any buffer subtracted results sorted against run time All_vsTemp contains all the primary analysis results sorted against temperature catter_vsTime contains the Scatter266 and Scatter473 and any buffer subtracted results sorted against temperature Fluo_vsTime contains the Scatter266 and Scatter473 and any buffer subtracted results sorted against temperature To create a custom analysis collection P Avacta age 7 ANALYTICAL Avacta Analytical Ltd 2012 AOOGAUNVH 4 ais m Q 1 lt gt Z gt ic lt n m 0 e Create the graph of the data you wish to add to the analysis collection o Select an abscissa typically Temperature for a thermal unfolding experiment or Run Time for an isothermal experiment o Select an ordinate Scatter266 and Scatter473 for SLS data at 266 nm and 473 nm respectively and the other parameters for fluorescence o Choose samples from the list under Select Results to Display or Add to a Collection This will disolay
95. guration The temperature will then increase by the chosen increment size indicated by Temp Step When the set temperature of the copper plate has been reached the instrument waits for the designated Temp Hold Time and then takes another set of measurements Measurements are taken at each temperature step until The Stop Temperature has been reached P 4 Avacta Seas ANALYTICAL Avacta Analytical Ltd 2012 File Ramp Log Normal Ramp Status Ramp Settings Temperature Data Current Temp Setpoint Temp Ramp Data Hold Time Current Step Step Count Q Edit 3 Apply x Cancel To alter the Ramp settings e Click amp EDIT e Type in a Start Temperature and Stop Temperature within the range from 15 C to 100 C e Type in the required Temperature Step Size C and Hold Time seconds e Click EJ APPLY to apply the changes Isothermal Settings For an isothermal type Experiment the temperature remains constant within a tolerance of less than half a degree over the full range of operating temperature Measurements are taken at each well and then the instrument waits for the designated Wait Period before beginning a second set of measurements This process is repeated for the number of times indicated by the Repeat Count To alter the Isothermal settings e Inthe Isothermal settings tab click BJ EDI e Type in the required Temperature between 15 C and 100 C Wait Period s
96. he method development section of this handbook Tm_Method uses an interpolation and a multiple Tm finding algorithm to determine multiple melting points with a sensitivity that varies depending on the intrinsic noise level of the data This gives an output TransTemp that contains multiple results sorted by significance The method also returns an uncertainty Tonset_Method uses sophisticated edge detection algorithms to calculate the onset temperature of either unfolding or aggregation lsothermal_Method fits a mono exponential curve to the data Viewing the table of results Finally go to Advanced Analysis Centre tab and the Secondary sub tab and ensure that RESULTS is selected OptimAnalysis Analyse Data OptimUtilities D Optim Advanced Data Analysis Engine v1 54 Development Aya cta Manager Advanced Analysis Centre Report Generator ANALYTICAL Primary Secondary Tertiary Method Developer Results for each Cuvette Run Well View Results of Primary Analysis Data Folder run000140 B1 Data Folder run000140 A1 Data Folder run000140 C1 Data Folder run000140 G1 Data Folder run000140 H1 Data Folder run000140 11 Select Display Mode _ DataFolder gt run000140 J1 EWG a Data Folder run000140 K1 Bar Chart Data Folder run000140 L1 Marker Plot Data Folder gt run000140 M1 e Data Folder gt run000140 N1 Data Folder run000140 01 Data Folder run0
97. hed data to reveal the points where the signal starts to increase most rapidly 4 Heaviside applies a Heaviside function to the differentiated data so that the cumulative signal level is analysed This removes problems that can be caused if a sudden onset is followed by a sudden decrease in signal 5 FindEdgeLoc finds the location of a positive going edge in the output of the Heaviside function The default is to find an edge that is 10 of the maximum intensity The sensitivity can be edited by changing the function s fraction parameter in the method developer For isothermal data where kinetic rate constants are required the isothermal_method will fit a mono exponential function to the data This will return the rate constant calculated from the data Creating and editing methods New methods can be created in a very simple manner using the Optim Analysis software The application of methods is grouped by analysis level in the software so methods are developed specifically for each stage of the analysis process To create a new or edit an existing analysis method you should navigate to the Advanced Analysis Centre tab and the Method Developer sub fab and ensure that EDIT is selected P 117 Avacta age ANALYTICAL Avacta Analytical Ltd 2012 QZ LU Y gt i lt Z lt Q LU ale Vat B O1 S AOOGAUNVH ais m Q F gt Z gt i lt n m 7 FS E Optim Advanced Da
98. idual sample wells to find an appropriate spectrum to subtract For this fo function correctly you need to have e Entered identical formulation information in the well in which the buffer will be loaded but leave the Sample Details column empty e Changed the reference column entry to TRUE This will generate output results which have an appended _B For example Ratio350_330 will be called Ratio350_330_B when a buffer soectrum has been subtracted Secondary Analysis As with primary analysis secondary analysis is performed by creating collections of inputs in this case primary analysis results Unfolding curves and SLS curves and applying analysis methods Secondary analysis looks at the time and or temperature dependence of the fluorescence or light scattering results fo obtain melting temperatures aggregation onset temperatures and kinetic rate constant P 2 Avacta age ANALYTICAL Avacta Analytical Ltd 2012 Retiontemtenie Analyse Data OptimUtilities D a Optim Advanced Data Analysis Engine v1 54 Development Ava cta ANALYTICAL Manager Advanced Analysis Centre Report Generator Primary Secondary Tertiary Method Developer View Results of Primary Analysis FEFELE TFT TTET IE ce EASE Te Oo S28 80000665 apa i Or Sseecne 2s ame ZUe Ff he A en E ES Results Choose One Abscissa E c o a z a a o 2 u e And up to Two Or
99. ific However the Administrator can also change Global data The list of users is an example of Global data The database contains a list of Users as created by the Administrator When logged in the Administrator can always edit and view the full list of Users Similarly the Companies Projects and Buffers and Additives sections contain Global data Samples and Experiment Runs are examples of Project specific data These lists only contain data that is specific to the chosen Project The following sections describe how the Administrator might use each tab to manage their data Companies This tab allows the Administrator to keep a record of the companies that are associated with an investigation for example companies that provide samples or experiment templates As a default Avacta Group PLC will be listed An important function of this list is to identify the source of bought samples in the database Please select a company from the list below A Sigma Aldrich Company Ltd New Q Edit T Delete To add a company e Click GS NEW e Fillin the name and details of the company in the window on the right e Click E SAVE This company will now be listed in a drop down list on the Samples tab so that their records can be kept on the database P Avacta Oe 2 ANALYTICAL Avacta Analytical Ltd 2012 a lt i 2 oS a O NN w Q Sy Ze OR SZ O O wal m Z lt IE 2g gt Z
100. ill not be saved to the database If you wish to save a copy of this soectrum you can export the data calibrated or raw to Excel using the E3 EXPORT button or by right clicking the spectrum in the list e To begin arun click L START RUN in the Run Control window All spectra collected during a run will be saved to the database automatically and can be accessed at any time by selecting the appropriate Project gt Experiment gt Run z iS a Ar oS LPO OFS ee FTA TE OLL aE PD p Alternatively select Start Run from the Acquisition gt menu or use the toolbar button View Spectra In this tab the user can view any of the spectra that have been acquired in the selected Run These spectra have already been calibrated using the default calibration procedures To view details of the calibration procedures see later in this manual 8 Load Export Graph Scaling C1 31 94 C 233 08s Min Intensity counts Max Intensity counts Min Wavelength nm Max Wavelength nm A Selected Spectra Min Intensity counts Max Intensity counts 65535 Min Wavelength nm 247 473307020489 347 47 397 47 Max Wavelength nm 503 80532404662 Wavelength nm Well No Meas No Setpoint Tem Actual Temp Setpoint Time Actual Time Setpoint Power Actual Power Time Stamp Name hoo a0 787 s000062 feiss sarazogooe o5 33 2010 20054 pmax 300e 36145 2 Jo 30 2a00x6asesz002 60 2o37 5
101. im applications These are described in further detail in the Method box on this tab and in Section XI on page 59 of this handbook a lt i 2 oS a O NN w Q Sy Ze OR SZ O O wal m Z lt IE e Choose an Experiment template by clicking once on one of the four options Stepped Thermal Unfolding Study To determine the formulation that provides the greatest resistance to thermally induced denaturation of proteins with increasing temperature steps Isothermal Unfolding and Aggregation Study To determine the formulation that provides the greatest resistance to thermally induced denaturation and aggregation of proteins at a constant elevated temperature over a period of time Stepped Thermal Unfolding and Aggregation Study To determine the formulation that provides the greatest resistance to thermally induced denaturation and aggregation of proteins with Increasing temperature steps Linear Thermal Unfolding and Aggregation Study To determine the formulation that provides the greatest resistance to thermally induced denaturation and aggregation of proteins with linearly increasing temperature e Inthe pop up box o Click NEW fo start a new Run o Or to view historical data choose a Run from the drop down list and click LOAD P Avacta ad ANALYTICAL Avacta Analytical Ltd 2012 ag gt Z U wo O O 7N INAWNALSNI ANY AAV MAOS LNANO The Optim software will now load the settings
102. import files from elsewhere on the hard drive by entering the file path details in the import window User Levels There are three levels of user in the Optim software 1 Analyst 2 Supervisor 3 Administrator The Analyst user level is a simplified user interface that has been designed to enable an operator to use Optim but restricts access to more advanced features An Analyst user can prepare and load samples into Optim take measurements analyse resulfs and generate reports A Project Supervisor has the same access level as an Analyst user except that they are able to create a new Project and manage the user access to that Project The Administrator user interface allows access to the user management area and includes advanced Data management in addition to all of the functions that are available to the Analyst and Supervisor users User tab access in Optim software The table below indicates which level of user can access each of the tabs in the current version of the Optim software Analyst Project Administrator Supervisor 1 Login a ee E aE 2 Users J SOTTO 3 Companies i pa lt S 4 Projects E a a E 5 Experiments 6 Samples 7 Runs 8 Formulations a ae ee a ee E NIN NIN YA 9 Measurements 10 View Spectra v v User has limited access to the features of this tab S Structure Overview The Administrator defines a user identity and assigns a list of projects to that user Within a
103. is results and initial primary slice results for wells that have a non zero replicate number defined in the sample P l Avacta age me ANALYTICAL Avacta Analytical Ltd 2012 QZ LU Y gt i lt Z lt oO O LU T HANDBOOK information If you have chosen replicates manually using this feature this will be where the data is collected e Duplicates all datasets with secondary analysis results or primary slice data but for wells that contain identical o Sample Name and concentration o PH o Buffer name and concentration o Additive names and concentrations e Debye Data contains all initial primary slice light scattering data and any secondary analysis results with the same units for example time average data where there is a variation in sample concentration There must also be at least one well containing a reference generally the buffer that the protein is dissolved in that has zero concentration of the buffer protein present and all other solvent characteristics the same and a solvent well containing just a single buffer and nothing else This solvent well may be the same as the reference depending on the particular formulation e Trend Data contains all data sets and data types that have a varying parameter So if there are samples with different concentrations of a particular excipient but all other conditions are the same then they will be grouped together in the collection AOOGAUNV
104. is section contains all the information you need to get started with the Optim 1000 Instrument and its accompanying software including how to power the instrument on and off safely The Optim software will already be installed on the PC Please note that the Optim Client software can only be supported on the PC connected to the Optim 1000 and should not be installed or copied onto any other computers In this part of the guide you will find information about the screen layout the user access levels and an overview of how the software manages data For quick reference Section VII has a quick start guide which summarises the main steps involved in making a measurement with Optim 1000 There is also a flowchart to demonstrate the typical decisions taken by a user during the measurement process A For safe operation of the instrument the user is advised to follow the instructions and guidelines given in this document A A N AN DANGER The user should NOT remove any of the panels on the instrument With the instrument turned on and the side panels removed there is a significant risk of exposure to potentially harmful laser radiation high voltage electricity and hot surfaces The service engineers may need to remove these panels whilst operating the instrument They should assess the Risks associated with the Hazards and take appropriate action to reduce these Risks to an acceptable level A set of risk assessments for the user ar
105. kgroundRange InoutData Spectrum to be acted upon OutputData Resultant Soectrum P 122 Avacta age ANALYTICAL Avacta Analytical Ltd 2012 RangeMin Minimum value of range RangeMax Maximum value of range This calculates the mean y value of the data in the specified range and subtracts this from all data points in the soectrum SubBackgroundRange2 InoutData Spectrum to be acted upon OutputData Resultant Soectrum RangeMinl Minimum value of range 1 RangeMax Maximum value of range 1 RangeMin2 Minimum value of range 2 RangeMax2 Maximum value of range 2 This fits a straight line to the data within the two specified ranges simultaneously and subtracts this line from the whole spectrum This is useful for example in calculating the integrated intensity of a peak above a sloping baseline where one uses this function with ranges either side of the desired peak then performs an integrate area function on the resultant Rescale InNoutData Spectrum to be acted upon OutputData Resultant Soectrum OffsetValue Value to Add fo all points this is performed before the multiplication ScalingFactor Value to Multiply to all points this is performed after the addition The following calculation is performed on each point in the input dataset Output Inout Offset ScalingFactor Absolute InoutData Spectrum to be acted upon OutputData Resultant Soectrum Absolute to force values to be positive 1 f
106. l Avastin in 0 M Histidine 0 68 5 69 283 0 061182 ZE 80 359 0 23853 O run000005 C1 1 mg mi Herceptin in O M Histidine 0 77 5 42 997 0 48634 O lt 68 382 0 088749 7 gt 79 985 0 18559 J run000005 Di 1 mg ml Remicade in 0 M Histidine 0 58 5 64 57 0 036161 81 101 0 20689 oO run000005 E1 1 mg ml Synagis in 0 M Histidine 0 T55 67 962 0 096526 z 80 633 0 088372 run000005 F1 1 mg ml Humira in 0 M Histidine 0 69 5 69 748 0 14202 80 902 0 065845 run000005 G1 1 mg ml Enbrel in 0 M Histidine 0 67 5 63 722 0 11221 79 309 0 073107 run000005 H1 41 mg ml Erbituxin 0 M Histidine 0 66 5 66 559 0 052084 83 183 0 73959 This data can then be copied to the clipboard by clicking COPY TABLE DATA The data can be exported to a comma separated variable file called SecondaryResults csv by clicking on S EXPORT ALL DATA The data can be added to the report by clicking e ADD RESULTS TO REPORT will add the data to the report but only those shown in the table and it will add each result in a separate table e ADD ALL RESULTS TO REPORT will add all of the results to the report adding each result in a separate table e ADD TABLE TO REPORT will add the data exactly as shown in the table into the report The secondary analysis results can also be represented in graphical form Selecting either Bar Chart or Marker Plot will disolay the results in this way To display results select a plot parameter such as TransTemp Tonset e
107. l then install the Optim Client 1 5 database You will not need to interact with the installer during this time Once completed a series of shortcuts will appear on your desktop These shortcuts will help you use the Optim Client software efficiently e Optim Client 1 5 this is the software itself Double clicking on this will start the software and begin communication with the instrument e Edit formulation this will allow you to edit the formulation information e Optim Manual this is the user manual for v1 5 of the Optim instrument e Optim Quick Start Guide a simple and quick way to get started using Optim e Export 1 5 where files exported from the client software are saved e Import 1 5 the source of formulation information for importing DO NOT USE THE FOLLOWING UNLESS DIRECTED TO BY AN AVACTA REPRESENTATIVE e Optim Reset 1 5 a tool used to restart sub Components of the Optim instrument e Database Config Tool a tool to change the default parameters in the instrument Ou i E Optim Client Optim Reset Optim Quick Import 1 5 2 1 5 Start Guide a EXport 15 Database Optim Formulati Config To Manual After this process has successfully completed and if you are upgrading from a previous software installation then double click on Optim_1_5_update exe to update the Optim instrument you are connecting to Optim_1_45_Update a i Optim_1_5 Update 1 0 0 0 Again you will need to enter th
108. le data will display a table view of this information that can be edited um Analysis Vie E E OptimAnalysis Analyse Data OptimUtilities x uma a x Optim Advanced Data Analysis Engine View amp Edit Sample Data OAvacta Sample Information Wel Sample_Ref Sample_Name Sample_Conc Sample_Units Sample pH Notes Replicate_No Analyse Reference Buffer_Name Buffer_Conc Buffer_Unit 1 mg mL Poly IgG in 0 02 MOPS M with 0 M GuHClatpH8 Poly IgG 0 TRUE FALSE 0 02 M QualificationData 1 mg mL Poly IgG in 0 02 MOPS M with 0 2 M GuHCl at pH Poly IgG TRUE FALSE 0 02 GuHCl study 1 mg mL Poly IgG in 0 02 MOPS M with 0 4 M GuHCl at pH Poly IgG TRUE 0 02 Protein_conc1 1 mg mL Poly IgG in 0 02 MOPS M with 0 6 M GuHCl at pH 2 Poly IgG TRUE 0 02 1 mg mL Poly IgG in 0 02 MOPS M with 0 8 M GuHCl at pH Poly IgG TRUE 0 02 1 mg mL Poly IgG in 0 02 MOPS M with 1 M GuHClatpH8 Poly IgG TRUE 0 02 1 mg mL Poly IgG in 0 02 MOPS M with 1 2 M GuHCl at pH Poly IgG TRUE 0 02 1 mg mL Poly IgG in 0 02 MOPS M with 1 4 M GuHCl at pH Poly IgG TRUE 0 02 1 mg mL Poly IgG in 0 02 MOPS M with 1 6 M GuHCl at pH Poly IgG TRUE 1 mg mL Poly IgG in 0 02 MOPS M with 1 8 M GuHCl at pH Poly IgG TRUE 1 mg mL Poly IgG in 0 02 MOPS M with 2M GuHClatpH38 Poly IgG TRUE 1 mg mL Poly IgG in 0 02 MOPS M with 2 2 M GuHCl at pH Poly IgG TRUE 1 mg mL Poly IgG in 0 02 MOPS M with 2 4 M GuHCl at pH Poly IgG TRUE 1 mg mL Poly IgG in 0 02 MOPS M with 2 6 M GuHCl at pH Poly
109. le to display a selection of data such as transition temperatures that may have variation in solvent conditions concentrations from well to well By selecting a dataset these are the primary analysis results including by default options such as Scatter266 for the 266 nm light scattering a further range of datatypes become available These data types include slices through the thermal unfolding isothermal data which will appear as options such as Temperature 15 C and RunTime 60 s and secondary analysis results such as TransTemp and Rate Constant etc Choosing combinations of these options and wells in the list box will disolay the graph on the screen changing the temperature or run time will change the particular slice that is being displayed Clicking gt ADD TO COLLECTION will add the displayed data to the collection Selecting items in the collection and clicking REMOVE FROM COLLECTION Once you have selected all the items that you wish to add to your collection you can SAVE COLLECTION to take forward to the analysis P 104 Avacta agen ANALYTICAL Avacta Analytical Ltd 2012 2355 H View Tertiary DataSets 260 ET SE SE SF TEA SE ST TE SEAE SE ee e Analyse 345 19 973 C nm Results FluoPeakPos Temperature a J1 K1 L1 M1 M OT P1 AZ B2 C2 D2 E2 F2 G2 H2 2 J2 K2 t2 M2 N2 02 Well Run run000172 View Tertiary DataSets Analys
110. le well that had the Reference option set to TRUE will be analysed and displayed so long as the Analyse option was also TRUE If any sample wells are present with Reference set to TRUE then the system will recognise this as buffer and will present an option to automatically subtract buffer soectra from sample spectra before calculating the primary analysis When this option is available you will see an option to enable buffer subtraction When this is checked and set to ON there will be an additional two methods available in the dropdown box P 2 Avacta aes ANALYTICAL Avacta Analytical Ltd 2012 Perform Analysis i v Analyse Spectra Choose a Method _ All Methods _ ScatterMethod 022 IntrinsicFluoMethod_022 ScatterMethod 01 ScatterMethod BufferSub IntrinsicFluoMethod_BufferSub ry i For the software to be able to subtract buffer soectra from sample spectra it is essential that when using the wizard all the relevant spectra are present within the same run When the analysis is completed you can choose which dataset you wish to display by selecting an X axis and a Y axis You can change the scaling of the graph by unchecking the Autoscale box and manually editing the minimum and maximum X and Y axis values The graph can be displayed in one of three modes 1 Markers the graph will be displayed with points on the graph 2 Lines the graph will be displayed with no points but with lines 3
111. lected Project will be listed here Any samples listed on this tab will also appear in a drop down list in the Formulations tab Please select a sample from the list below O N acetyl L tryptohpanamide here is a description O N acetyl L tyrosinamide Here is another description To add a new Sample to a Project e Click NEW e In the window on the right type in a sample Name ID and brief Description These will appear in the list of Samples on this page and in the drop down menu on the Formulations tab Note The description field has a character limit and wil be highlighted in red if the description is too short Avacta rege ANALYTICAL Avacta Analytical Ltd 2012 e All other information is optional You can record the Lot No Concentration Appearance Molecular Weight and Expiry date in this window e f your sample has been supplied or manufactured by another company select the appropriate companies from the drop down lists at the bottom of the window the list of companies will be managed by the Administrator Click G SAVE Any Samples associated with a given Project will be listed by Name in the Formulations tab drop down list which will be discussed later Experiments The user is provided with four templates for experiments listed below Within a Project any or all of the experiments can be performed Each of the templates has predefined settings that are suitable for typical Opt
112. lue Std Dev Units All_vsTemperature Well C1 Tonset_Method_153 TransTemp 59 621 0 018114 C Scatter_vsTemperature Analyse Isothermal_Method_153 Fluo_vsTemperature Plot BarycenFluo All_vsRunTime vs Temperature Scatter_vsRunTime ae Saaaa Ready To analyse this data e Select the Analysis Collection that you wish to analyse in this case MyAnalysisCollection is selected Saved Analysis Collections All vsTime scatter_vsTime Fluo _vsTime All vs Temp scatter vsTemp Fluo vsTemp MyAnalysisCollection e Select the methods you wish to use typically select Tm_Method for fluorescence data vs temperature Tonset_Method for scattering data vs temperature and fluorescence data to get the onset of unfolding and lsothermal_Method for collections which include a variation with time P Avacta ngea ANALYTICAL Avacta Analytical Ltd 2012 Analysis Method Tm_Method_022 Tonset_Method_022 e To analyse the trace on screen click Y ANALYSE e View the result as a line on the graph and match the colour with the value displayed in the Results box BarycenFluo nm QZ LU Y gt i lt Z lt Q O LU T Vath B 1O1 S TransTemp 64 57 61 101 0 20689 e lf the method has resulted in an error in the calculation you can select the relevant result by clicking on it in the graph then o Drag it to the location where you wish it to be right click and sele
113. m U gt re gt Z Pulse width Wavelength Power output j wi Laser class Repetition rate Danger to Eyes AN DANGER Laser Hazard Direct or indirect exposure of a laser beam to the eyes may result in permanent damage to the eyes including photokeratitis photochemical cataracts and retinal injuries If operating the instrument without its covers always wear appropriate laser protective eyewear Hazard to Skin AA Physical Injury Hazard Direct exposure of a laser beam to the skin may cause erythema melanoma accelerated skin aging increased pigmentation and photosensitive reactions If operating the instrument without its covers always wear appropriate personal protective equipment gloves Laser Warning Labels CAUTION LASER RADIATION WHEN OPEN This label is displayed on the top of each of AND INTERLOCKS DEFEATED DO NOT STARE INTO DEAM OR VIEW the left and right side panels of the CIRECTLY WITH OPTICAL INSTRUMENTS l AVOID EXPOSURE TO BEAM instrument AVOID EVE OR SKIN EXPOSURE TO DIRECT OR SCATTERED RADIATION Physical Hazards Moving Parts Physical Injury Hazard Moving parts can crush and cut Keep hands clear of moving parts including the motorised door while servicing or operating the instrument Hot Plate oe Physical Injury Hazard The plate where the samples are located may become hot during operation Allow sufficient time for it to cool before moving or ch
114. ment e Start up the Optim 1000 Client software by double clicking the link on the PC desktop the Login tab should be displayed The software cannot be run when the Optim instrument is switched off a lt i 2 oS a O NN w Q Sy Ze OR SZ O O wal m Z lt IE Login The Analyst s login details will be set up by the Administrator The Administrator or Project Supervisor also has control over which projects are available to Analyst Users User Login Username Password Initial Project Status logged out e Insert your username and password e Choose a Project from the drop down list e Click LOGIN Projects To view the description of a Project or to change to another Project after you have logged in use the Projects tab P Avacta age ANALYTICAL Avacta Analytical Ltd 2012 Please select a project from the list below g New Project g New Project2 e Inthe list on the left click on the Project you wish to work on All the settings and data for that Project will then be loaded aE gt Z U W O G 7N INAWNALSNI ANY FAV MEOS INID If you have been assigned the responsibility of being a Project Supervisor you can edit the details of the Project You also have permission to create a new Project Samples This tab contains information about the samples that will be investigated as part of the selected Project Only samples relevant to the se
115. mium level for peak TmName Name of Results File This function calculates the differential of the InoutData and looks for a peak in this data between the desired ranges P 12 Avacta Boe tee ANALYTICAL Avacta Analytical Ltd 2012 FindMultpleTm InoutData Spectrum to be acted upon RangeMin Minimum value of range RangeMax Maximum value of range SmoothWidth DataPoints for Median Smooth and Averaging SignalToNoiseMinimium level for peak MaxNumPeaks Maximium number of peaks TmName Name of Results File This function calculates the differential of the InoutData and searches for peaks within the given range It estimates the uncertainty in these Tm by estimating the local noise at these points Onset InoutData Spectrum to be acted upon StartPoints Number of points to start search Threshold S N threshold Sensitivity Chi squared multiplier before onset deemed valid TonsetName Name of Results File This function tries fo determine an Onset point by determining the point beyond which the data no longer fit to a straight line MatchCharacteristic InoutData Spectrum to be acted upon AnalysisType Class of Analysis ListName Name of List of Data to search for match OutputData Resultant CharAbsName Name of Abscissa Characteristic for closest match If empty then this parameter is ignored CharAbsValue Value Characteristic If empty then the value is taken from InputData CharName Name of Charact
116. mpleMaths Interpolate FindMinMaxLoc FindEdgeLoc IntegrateArea IntensityRatia BarycenticMean PeakFit ExpDecayFit simpleTm FindPeakTm FindMultple Tm Onset MatchCharacteristic S O O O O O E E HR E E E E E E E E E B E E E E Clicking RESET will reset the selections to the Avacta Analytical supplied options As discussed in the previous section the instrument obtains different parameters that can be plotted against analysis results to show trends By default only the actual measured temperature and the time after the start of the experiment are available for display If you wish to change the available parameters you can do so in the ANALYSIS settings P Avacta aCe ANALYTICAL Avacta Analytical Ltd 2012 QZ LO Y gt lt A lt 2 A O Lu ale HANDBOOK AOOAUNVH dASAIVNV WILldO JHL Secondary Analysis Abscissa actualTemperature actualTime setpointTemperature timestamp Before you start adjusting verifying the sample formulation During the pre run configuration in the client software information can be entered that defines the formulation conditions for each of the sample measured If this information is not supplied or is entered incorrectly it can lead to confusion during analysis or not being able to perform tertiary analysis It is possible to view and edit this information in the Optim Analyser software Selecting the Optim Utilities menu and selecting View and Edit samp
117. n using the Excel import template it is possible to enter up to three additives per sample well If you wish to use more you must add them using the manual method outlined above e For each sample select an entry for the Sample Name using the dropdown box at the side of each cell and complete the rest of the formulation information e lf you wish to perform a buffer background measurement for example if you have fluorescing Compounds in your buffer the analysis software P 4 Avacta adie ANALYTICAL Avacta Analytical Ltd 2012 will attempt to find the spectra for the relevant buffer and subtract it from the sample spectra automatically To do this o Enter identical formulation information in the well in which the buffer will be loaded but leave the Sample Details column empty Change the reference column entry to TRUE Changing the Analyse column entry to FALSE will not affect the buffer subtraction but the buffer soectra will not be analysed It will not be possible to see how the buffer fluorescence and light scattering signals change with temperature e lf you are measuring replicates insert a value into the Replicate No column all rows with the same replicate number will be grouped together as replicates in the Optim Analysis software e Type in a filename and click CREATE FORMULATION A file will be created in the Import folder e In the software go to the Import Export box and input the Excel file name and locati
118. ne the formulation that provides the greatest resistance to thermally induced denaturation and aggregation of proteins with increasing temperature P Avacta ad ANALYTICAL Avacta Analytical Ltd 2012 Method Examining the intrinsic fluorescence and UV and blue scattered light while stepping up the temperature of the sample When under thermal stress the proteins begin to unfold and aggregate This is indicated by an increase in the intensity of the scattered light and changes in the fluorescence spectra By using a UV laser for high sensitivity measurements and a higher power blue laser with a broader focus aggregate size differentiation can be achieved The sample is heated using a stepped profile All the samples are equilibrated at the measurement temperature and then held at this temperature while they are measured The temperature is then incremented the samples are equilibrated held and measured again 4 Linear Thermal Unfolding and Aggregation Study Aim To determine the formulation that provides the greatest resistance to thermally induced denaturation and aggregation of proteins with linearly increasing temperature Method Examining the intrinsic fluorescence and UV and blue scattered light while linearly increasing the temperature of the sample When under thermal stress the proteins begin to unfold and aggregate This is indicated by an increase in the intensity of the scattered light and changes in the fluorescence
119. nsicFluoMethod ScatterMethod_BufferSub SampleBufferSpectra _ None _ IntrinsicFluoMethod_BufferSub Secondary analysis methods The default secondary analysis method includes a method fo find the onset of the aggregation temperature and up to four melting points in the unfolding data These can be edited as required or used as templates for the user s own methods The secondary analysis method that is used to determine transition temperatures or melting temperatures if you are referring to thermal Unfolding temperatures is called Tm_method The method consists of two functions that occur in order 1 Interoolate to fill in any gaps in the primary analysis results data so that even if a low temperature resolution scan is performed it will find a result that is the most accurate value 2 FindMultipleTm this calculates the differential of the interpolated data and finds the position of peaks that exceed a specified signal to noise level of the smoothed differential to give the points of inflection in the curves The sensitivity can be changed in the Method Development tab by changing either the smooth width or the signal to noise threshold An uncertainty is estimated by using the method described here The local noise of the unfolding curve parameter versus temperature is estimated by calculating the residual between a running a local smooth and the original data calculating the local standard deviation
120. nstrument is classified as portable equipment i Electrical Shock Hazard Severe electric shock can result from operating the instrument without its panels in place Do not remove the instrument panels High voltage contacts are exposed when the instrument panels are not in place Z O lt o lt o FE a A Power A OANGE Electrical Hazard Use properly configured and approved line cords for the local voltage supply ADANCI Electrical Hazard Continuous grounding circuitry is vital for safe operation of equipment Never operate the instrument with grounding conductors disconnected AS Electrical Hazard Plug the system into a properly grounded receptacle with adequate current capacity Fuses A Fire Hazard Improper fuses or high voltage supply can damage the instrument wiring system and cause a fire Before turning on the instrument verify that the fuses are properly installed and that the instrument voltage matches the local power supply i Fire Hazard Replace fuses only with fuses of the type and rating specified for the instrument Laser Safety A caution Laser Hazard Use of controls or adjustments or performance of procedures other than those specified herein may result in hazardous radiation exposure Optim is a Class laser product The lasers within the instrument have the following specifications P l Avacta eget ANALYTICAL Avacta Analytical Ltd 2012 8
121. o do so Clicking on one of the target buttons will toggle between hiding and displaying all wells in the selected MCA In the example shown wells D1 D2 D3 L1 L2 and L3 had ANALYSE set to FALSE in the formulation information that was set within the client software and are thus not present in the analysed data To export the primary analysis results click EXPORT If you select All Wells then the software will export all the data from all the wells of the primary analysis that is currently being viewed If you select Displayed then it will export only the displayed graph data Selecting All Data will export all wells for all primary analysis results To add the primary analysis results to the report click ADD TO REPORT Again by selecting All then all the data will be added to the report in the Primary Analysis section By selecting Displayed then only the displayed graph will be added to the report When you have finished the primary analysis click GOTO SECONDARY ANALYSIS gt to proceed onto the next level of analysis If you wish to change the run that you are analysing within the same study you can do so by making the selection on the primary analysis screen You can also switch between studies by choosing a study from the dropdown menu To add new run data to the software or add or load another study you will need to click BACK TO LOAD RUNS to do this Performing Secondary Analysis After the c
122. of GUHCI Smoothing the data can make the graphs look smoother hmm P Avacta Se ee ANALYTICAL Avacta Analytical Ltd 2012 Vath B 1O1 S AOOGAUNVH 4 ais m O me gt Z gt P lt n m 0 FluoPeakPos nm 20 40 60 80 100 Temperature C Selecting one of these will display a slider such that the width of the smooth can be selected The larger the smooth width the lower the resolution of the curves and there Is a potential to artificially remove smaller less significant transitions from the data If in doubt a good default choice is a 3 point binomial smooth this will present the data well without introducing significant artefacts Clicking on d dx will present the data as a differential of the unfolding curves There are options to display the differential before it is smoothed or after it is smoothed Displaying the differential unsmoothed is not recommended as this is always noisy due to small fluctuations in the signal being amplified in a differential view The top graph below represents an unsmoothed differential and the bottom graph has a three point the minimum possible smoothing applied P 9 Avacta age 72 ANALYTICAL Avacta Analytical Ltd 2012 Nea g AK N a ATAN N I I WAM i NRS hare il RAYAN Ns AN d FluoPeakPos d Temp nm C Temperature C d FluoPeakPos d Temp nm C Temperature C This dat
123. of appropriate moving equipment and proper lifting techniques Who might be Service engineer other helpers inappropriate lifting harmed and techniques may result in a painful back or other how musculoskeletal disorder What is being The service engineer is trained in proper lifting techniques done already Where assistance is required from others they should also have received appropriate training With the use of proper techniques the risk is very low What further If assistance is required from others the service engineer should action is ensure they are properly trained With the use of proper necessary techniques the risk is very low By whom Wheng P l Avacta age tee ANALYTICAL Avacta Analytical Ltd 2012 XVIII GLOSSARY OF TERMS Physical things Micro Cuvette Array MCA The black blue anodised sample holder that contains an array of 16 square or round quartz micro cuvettes Cuvette Holder the central black unit into which the micro cuvettes round or square are inserted The cuvette holder has one or two parts depending on the cuvette type Frame blue the frame is made of two hinged caps each of which will hold a rubber seal The cuvette holder slides into the holding cap of the blue anodised frame and Is held in place by the closing cap and clip Cuvette A round or square micro cuvette Well An alternative name for cuvette used for descriptive purposes to rep
124. of these residuals and correcting for the bias caused by only taking local samples This has been verified to be correct with simulations The gradient of the unfolding curve at this point is determined by the height of the first differential P 11 Avacta ego ANALYTICAL Avacta Analytical Ltd 2012 The uncertainty in the transition temperature is then estimated as noise gradient The data is not fitted to any particular function so determining the uncertainty in a fit parameter from a chi squared or similar statistic is not possible If an attempt were made to fit the data to a particular functional form then theoretically the error in certain fit parameters Could be very well determined but this is only true if the functional form were a true representation of the shape of the data which in practice is rarely the case The secondary analysis method that determines the onset temperatures whether the onset of Unfolding or Tagg the onset of aggregation is more complicated It consists of five functions 1 Interoolate to fill in any gaps in the primary analysis results data so that even if a low temperature resolution scan is performed it will find a result that is the most accurate value 2 Smooth performs a five point median smooth of the interpolated data to reduce the effect of spikes in the data due to dust etc causing an artifactual anomalous result 3 Differentiate differentiates the smoot
125. omial Savitzky Golay weighted means End effect Bounce OutputData Resultant This function performs a running Smooth of the input data soectrum Heavyside InNoutData Spectrum to be acted upon Polarity Positive running maximum Negative running minimum Auto function tries to work out which way the trend Is going OutputData Resultant AutoPolIName Name of the output value will 1 for positive or O for negative This function performs a Heaviside operation on input data soectrum Each data point in the output determined from all points in the input up to the point under consideration For a positive polarity each value of the output is the maximum of alll previous values EstimateNolse InoutData Spectrum to be acted upon SmthPnt Number of data points to use for initial smooth StdDevPnts Number of data points to use for standard deviation OutputData Resultant This function estimates the noise at each point in the InoutData It performs a running smooth then calculates the residual between this smoothed dataset and the original dataset It then calculates the local standard deviation of these residuals and corrects this for the size of the sample to give an estimate of the local standard deviation at each point SimpleMaths InoutData Spectrum to be acted upon AuxinputData Spectrum to be added subtracted eg a buffer spectrum multiplied or divided denominator OutputData Resultant Function InoutData
126. ompletion of primary analysis clicking on ANALYSE RESULTS will carry out secondary analysis By default the software will use the default secondary analysis methods and auto detect the type of experiment carried out If you wish to use a different analysis method such as one defined and saved by an advanced user you can select this in the dropdown menu Perform Analysis v Analyse Results Choose a Method _ All Temperature Methods _ When the analysis is completed you can choose which parameter for example transition temperature or onset temperature from a particular dataset that you wish to display by selecting them from the dropdown boxes You can display the data either by well reference such as Al B1 etc by the sample name such as Poly IgG or Lysozyme or the sample details which is the full information P 4 Avacta age ANALYTICAL Avacta Analytical Ltd 2012 You can change the scaling of the graph by unchecking the Autoscale box and manually editing the minimum and maximum Y axis values The graph can be displayed in one of three modes 1 Bar Chart the graph will be displayed as a bar chart with the parameter indicated by the magnitude of the bar In the case of multiple values each well will have multiple bars as shown below The most significant result will be shown in the graph first 140 f 120 100 TransTemp BarycenFluo a4 LUI Y gt lt Z lt 2 OW O
127. on e Click IMPORT ag gt Z U wo O O 7N INAWNALSNI ANY FAV MAOS LNANO An example formulation editor sheet is shown below ils Sample Sampie Sample Replicate Realise Reference Buer Name Buffer caa Adate 1 Name Additive1 gnilo Nota Con Units pH No Conc Units Conc 1 Units m TRUE FALSE Tribasic Sodium Phosphate 0 1 NV vi TRUE FALSE Tribasic Sodium Phosphate 0 1 NV TRUE FALSE Tribasic Sodium Phosphate 0 1 M TRUE FALSE Tribasic Sodium Phosphate 0 1 NV TRUE FALSE Histidine 0 03 NV TRUE FALSE Histidine 0 03 M TRUE FALSE Histidine 0 03 Nv TRUE FALSE Histidine 0 03 N vl TRUE FALSE Histidine 0 05 M NaCl 0 01 M TRUE FALSE Histidine 0 05 N NaCl 0 02 TRUE FALSE Histidine 0 05 NV NaCl 0 01 N TRUE FALSE Histidine 0 05 N Nacli 0 02 M FALSE TRUE Tribasic Sodium Phosphate 0 1 NV 1 FALSE TRUE Histidine 0 03 N vi TRUE TRUE Histidine 0 05 M Nacli 0 01 M TRUE TRUE Histidine 0 05 N Nacli 0 021M Pwr wWNeEN BF C N c 3 a o H Ls aD PWN NIN TOTP WP WwW Ph WwW Pb Ww Ww gt gt Project samples Create Formulation 3 iE gt dy BG 85 C In this case there are two sample molecules Rituzimab sic and Trastuzumab The following points are worth noting about this formulation 1 There are four duplicates the first eight formulations a Aland Cl b Bl and D cC El and G d Fl and H 2 There are four buffers being measured a M1 is the buffer for Al and Cl and at each temperat
128. on select Start in the method generator and SubBackgroundRange in the list of functions Click INSERT FUNCTION INTO METHOD to add this function to the top of the list in the method This function will take the input spectra subtract an average of the signal in the range specified by the RangeMin and RangeMax parameters and create an output wave in a different dataset RangeMin and RangeMax can be changed by typing different values in the parameter editor or selecting the line in the preview area dragging it to a new position right clicking and selecting Sef When a parameter has a checkbox next to it ticking the box will display the parameter in the preview area in the colour specified So RangeMin will be displayed as a blue line and RangeMax as a green line Parameters SubBackgroaundF ange Show Parameter lnputD ata OutputD ata RangeMin Rangel ax Datasets in the Optim Analysis software are called Dx where x is a number starting at O for the highest level dataset All the functions that are currently in the method act on data that is in the dataset DO the results of the function become dataset D1 In P 11 Avacta age hie ANALYTICAL Avacta Analytical Ltd 2012 QZ LU Y gt i lt Z lt Q LU ale Vath B 1O1 S AOOGAUNVH ais m Q F gt Z gt i lt n m 7 the method generator select IntensityRatio and click EDIT FUNCTION PARAMETERS In the function
129. or example Project Experiment Run If is advisable to look through the Glossary before beginning to use the instrument Index To find information relating to a particular tab or topic refer to the Index at the end of this guide Instrument handlin Watch out for the following alert words in the text they will advise you of a physical hazard or an important steo for you to carry out The alert words indicate the particular level of observation or action as defined below re Indicates information that is necessary for proper instrument operation or sate usage P Avacta oe ANALYTICAL Avacta Analytical Ltd 2012 L CATION Indicates a potentially hazardous situation that if not avoided may result in minor or moderate injury It may also alert against unsafe practices LANANG Indicates a potentially hazardous situation that if not avoided could result in death or serious injury DANCER Indicates an imminently hazardous situation that if not avoided will result in death or serious injury This is limited to only the most extreme situations P Avacta Se ANALYTICAL Avacta Analytical Ltd 2012 PREPARATION I SITE PREPARATION This section of the manual describes how to prepare your site for installation of the Optim 1000 and how to get the instrument up and running There are important safety warnings included here which must be observed by all personnel that have access fo
130. or negative This function takes each y value of the input data and converts it to the Absolute i e the sign of the value is forced to be either positive or negative depending upon the value of Absolute of this value in the output dataset Sign InoutData Spectrum to be acted upon OutputData Resultant Soectrum This function takes each y value of the input data and converts it to the Sign i e 1 if negative 1 if not negative of this value in the output dataset Integrate InoutData Spectrum to be acted upon OutputData Resultant Soectrum This function performs a numeric integration of input data soectrum The first data point of the OutputData has value zero and there is one more data point in the OutputData than in the InoutData P 12 Avacta Se a ANALYTICAL Avacta Analytical Ltd 2012 QZ LU Y gt i lt Z lt Q LU ale Vath B 1O1 S AOOGAUNVH 4 ais m Q me gt Z gt i lt n m 0 Differentiate InoutData Spectrum to be acted upon OutputData Resultant Soectrum This function performs a numeric differentiation of input data spectrum There is one less data point in the OutputData than in the InoutData Smooth InoutData Spectrum to be acted upon SmthPnt Number of data points to use for the smooth Must be Odd will round down to odd if not and minimum value is 3 Method Smooth Method options Box simple mean Median middle Bin
131. or neither Calibrations When data is captured using the program four types of calibration are applied to the raw camera capture data before the spectra are displayed in the View Spectra tab It is possible to export to data from this tab to Excel without these calibrations by choosing Export Raw Data from the menu options The four calibrations are applied in the following order 1 X axis Calibration Converting pixel numbers into an appropriate wavelength scale 2 Background Subtraction Subtracting a CCD camera imposed background so that zero intensity means no photons are hitting the camera 3 Device Response Calibration Correcting for the variation in efficiency of the CCD s response to a range of wavelengths so that the intensity of the soectrum Is proportional to the number of photons 4 Laser Power Calibration Calibrating the laser power over the full set of measurements to account for small variations in power over long time periods Of the four calibrations performed two are automatic and two are pre calibrated Functions 1 and 3 are configured by Avacta on installation and checked at every subsequent service The four calibration procedures are explained in greater detail below 1 X axis Calibration The CCD detector is an array of pixels When a photon is incident upon a pixel it causes a volfage change across the pixel The soectrograph uses a grating to spatially separate light of different wavelengths by
132. ou can insert new functions or edit existing functions How to select data for Method Development e Primary o To display a particular soectrum in the method developer then you should select it from the Select Spectra list in the Primary sub tab of the Advanced Analysis Centre highlight the desired spectrum so that it Page 11 Avacta age ll ANALYTICAL Avacta Analytical Ltd 2012 appears in the graph and ensure that no spectra collections are highlighted o Alternatively you can select multiple spectra or a spectra collection and the first soectrum of this group will be displayed in the preview area of the Edit tab The rest of the selected spectra can be scrolled through using the same method in the Test tab e Secondary o The first dataset of the selected Analysis Collection will be displayed in the Edit tab The remaining datasets can be scrolled through in the Test tab Editing the default primary analysis fluorescence method to include a background subtraction Follow this example Load the IntrinsicFluoMethod primary analysis method into the method developer You will see that there are four functions as part of this method as discussed in the previous section NB When spectra are acquired in the client software the instrument automatically subtracts a dark count For the purposes of this tutorial the example dataset has spectra which have not had the dark count subtracted To add a background subtracti
133. posal provisions to reduce the environmental impact of waste electrical and electronic equipment WEEE e fthe Optim unit is removed for disposal the user should dispose of it in an appropriate manner The Optim unit is made primarily from standard metal plastic and electronic components The WEEE regulations allow the onus for disposal of such equipment to reside with the user The MCA sample holders may contain hazardous samples if such samples have been run by the user The user should follow appropriate local and national regulations for their disposal P l Avacta eget ANALYTICAL Avacta Analytical Ltd 2012 IIl SOFTWARE INSTALLATION AND ACTIVATION Your software The software Is supplied in a package as shown below The package contains two USB compatible devices one which contains the installers for the software along with some documentation The other is a security dongle to prevent unauthorised copying or distribution of the software Z O s lt o lt o FE a A Each dongle is initially supplied in demo only mode and must be registered with Avacta Analytical before use The software can be used for one month If the software is not registered after this time then it will not be possible to use the software To register and activate the software contact Avacta Analytical directly by emailing optim avacta com supplying the serial number of your instrument the date of installation and who
134. r Additive to the database use the Samples tab In the Samples tab click Gd NEW BUFFER A window will pop up prompting you to type in the name and details of the new Buffer Avacta Analytical Ltd 2012 OAyacta Page 56 ANALYTICAL e Click E SAVE to add the new buffer to the database This buffer wil now appear in the buffer drop down list on the Formulations tab e To input a new Additive to the database click NEW ADDITIVE and follow the same procedure a lt i 2 oS a O NN w Q Sy Ze OR SZ O O wal QO Z lt IE P Avacta age ANALYTICAL Avacta Analytical Ltd 2012 XI EXPERIMENT SETTINGS This section contains the details of all of the preset functions in the software Here you can find definitions of the different types of Experiment discussion of the temperature step function explanations of the calibrations that are applied to the data and information about the analysis macros that are performed at primary and secondary level Experiments The user has been provided with four templates for experiments listed here Within a Project any or all of the experiments can be performed Each of the templates has predefined settings that are suitable for a typical stability investigation These are described below The capability to create user defined experiments will be included in a future release of the software a lt i 2 oS a O NN 5 Z w Q TA O SZ O wal m Z
135. r in the advanced mode is logged The Analysis Log sub tab enables you to view the log of actions that have been performed in the study and in the current session Report Generator Selected Log OpAn_Fref SetDataPath OpAnDP Studies VAVACTASERVER RedirectedFolc OpAnFile_StudyLoadStudy Data H 30 12 2011 08 23 OpAnHist_ StudyLoadHistory OA Study0 ff 30 12 2011 08 23 OpAnRept_StudyLoadRepornt OA Study0 Mf 30 12 2011 08 23 OpAnPim UpdateSpecData ff 30 12 2011 08 23 OpAnPrim_ SpecMakeDefaults run000005 Mf 30 12 2011 08 23 OpAnPrim_RunAnalysis PSL1 ScatterMethod 022 V 30 12 2011 06 25 QOpAnPrim RunAnalysis PSL1 IntrinsicFluoMethod 022 ff S09 2 2011 0 OpAnSecd UpdateSecdData runQ00005 ff 30 12 2011 06 24 OpAnSecd AnaListMakeDefaults runQ00005 A 30 12 2011 06225 OpAnsSecd AnaListMakeDefaults runQ00005 A 30 12 2011 00 25 Minnaard Anal icthdakaNafaulteMninhihihinina s ff AANA NA IT Settings A variety of settings can be configured in the Settings sub tab There are three classes of settings GENERAL settings for miscellaneous settings LOAD DATA settings for setting the file paths for loading data and selecting which parameters to extract from the spectra ANALYSE settings to configure the analysis engine In GENERAL settings it is possible to edit the colours that are used to generate plots and graphs The default colour scheme is a colour table featuring 48 different colours each comprised of a valu
136. r requirements a In case of emergency you must be able to immediately disconnect the main power supply to the instrument The main Optim 1000 unit is factory configured to operate interchangeably at 120 VAC or 240 VAC Voltage Frequency Current drawn for Fuse Rating Max Power Power consumed Z O lt o lt o FE a A 220 240V 50 60 Hz 0 5 1 A 0 15 KW VA 110 125 V 50 60 Hz 1 25 2 5A 0 15 kW VA Over current protection gt 110 Checklists Check that the location fulfils the following criteria MI The bench or table area is sufficiently large and can withstand the weights of the instrument MI There are sufficient mains power sockets within range M The location of the instrument and computer allows for appropriate ergonomic usage Check that all the following components are present M Optim 1000 analytical instrument Mains power lead USB lead Ethernet lead Desktop PC N WN WN WN AW Monitor VI Mouse If your installation site is suitable and all of the Optim 1000 components are present you are almost ready to begin Your installation Engineer will set up the instrument for you so in the meantime take the time to read the important safety information in P 9 Avacta Sa ANALYTICAL Avacta Analytical Ltd 2012 the following section so that you can look out for those potential hazards when you begin to operate the instrument 8 m 23 gt re gt O Z
137. reated analysis studies If you have added a new method to the default methods you can click WSAVE DEFAULT METHODS and it will appear in all new studies If at any point you wish to restore the user default methods you can click 7LOAD USER DEFAULT METHODS To restore the default methods to the factory settings click RESTORE FACTORY METHODS The factory default methods are hard coded into the analysis software so as new versions are released and new methods added they can be added to your studies by restoring factory methods and manually copying them across to your studies Definition of standard functions included in the Optim Analysis software In this part of the handbook you will find a definition of all functions and their parameters that are included as standard with the Optim Analysis software For any given Method the input dataset is labelled DO This can be a spectrum temperature unfolding curve or anything else A Function can take this DO as input and other parameters and output either a new dataset say D1 and or some characteristic values or Results Subsequent Functions can take any of the datasets as input i e DO or D1 etc and other parameters some of which may be previously determined characteristic values from within the same Method Finally a list of the Characteristic values or results determines results to be stored as Output from this Method The following Is a list of available Functions SubBac
138. resent the chamber that contains the formulation Sample The protein or primary molecule of interest Formulation The mixture of sample with buffer and optional additives which is inside a cuvette or well of the MCA Hot Plate The copper plate with brass spigots to mount MCAs The plate heats the samples Stage The hot plate is mounted upon a stage The stage moves by motor control or as a manual drawer Software things Capture A single shot of the CCD camera a raw image that can be processed to produce a spectrum Single Shot This command fells the camera to take a capture using the current settings This capture will not be saved to the database Run A succession of captures with user defined settings automatically saved to the database Measurement A measurement that can be performed on the sample e g UV fluorescence or blue light scattering Measurements are defined by the choice of experiment Experiment A template for a standard operating procedure SOP that defines the combination of measurements that will be performed on the sample OAvacta Avacta Analytical Ltd 2012 Page 136 Project The user selects a project at the Login stage of the Client Software This gives them access to all raw spectra analysis results and reports for a given study Generally a new Project is created for each molecule of interest Study A folder of work inside the Analysis Software where
139. s 65 PP TIN STOO Ke a erter era canersen ANE EAEAN EE AEA EEE A EEEE AE 65 AN TECA ea E A EE EEO 66 Choosing the analysis mode you Want TO USEC e ussssssssesresresresresresereresrreresreeeesersees 68 Alle THE OPTIM ANALYSIS PRLOSOPHY perrera EENET ESEA EE EES 69 AIV THE OPIMANALYSIS WIZARD creas tencatencarsucnncniaetuaassrostenstaneveatbensbenttosieuthacaenauends 7 Loading data into the WZ Ol Oa ncteiaccuctcandasteanscacesrnsateoniasitesdinsdtncaiasitecdinsiecnasiteciaen 7 Penormng PINAY ANGIV S19 ascris tas scancclacostcesiastuesiacostcssiastuestacesessnacad 72 Performing Secondary AndadlysisS sxnaces san siacciiastannthiatamnbiaoaensindasabsinoslovsindusaaioosloseinieesead 74 XV ADVANCED DATA ANALYSIS MODE antec neiaaennceneencatanacosecsbeansanttaseladssacanenseenes 8 Managing the software and Loading data ee ccccssecescceseccesccesccescensceusecuseeuss 8 FAV PVCU SIS ices ceases oor E AEA E E EAE E E EE 86 S CONA A SIG ser stent e EEA E OENE EEE 92 Generating a sample repO0rft eessssseesesresresresrerressessessessesseseesseseesseseeseeseesseseeseeseess 105 XVI EDITING AND DEVELOPMENT OF ANALYSIS METHODS ccc ccccceece esse eeseeeees 115 SCS TOU TIS VO Sag src escceet E E E E EEE E ES 115 Creating and SANA WONG car giecttartyena tiene nuiedtiudened Gaundeuesasnsatenannsaussadedincnundounsassiics 117 MOITO G SC wee eres E de ewaren E EE EA E 12 Definition of standard functions included in the Optim Analysis software
140. s that are available to load Select those that you wish to load to select multiple runs hold down shift and click LOAD The system will then load sort and automatically classify each of the spectra according to the information present in the formulation file P 1 Avacta age ANALYTICAL Avacta Analytical Ltd 2012 AOOGAUNVH a m O mA gt Z gt a lt 2 m 7 Optim Analysis Add Data Add Data to Study MyNewStudy Available Run Data Once loading of the data is completed the Files sub tab will display the data that is currently loaded into the study Study Study Data ApplicationNote e alm Application ote a No Spectra Sample Info rundOooc4 2452 a To add further data simply click in the Study Data section The table below describes the functionality of the local buttons in the Study section of the files tab Create a new study Open a saved study Save the currently open study Remove the current study from the software note this does not remove the study from the file system if can still be loaded P 2 Avacta age ANALYTICAL Avacta Analytical Ltd 2012 L ONINCONANI SAVE THE STUDY AT THIS STAGE OR YOU WILL HAVE TO RELOAD AND REANALYSE THE DATA YD p You can also save the study by clicking the orange Save button on the El toolbar The Analysis log Every action performed in the Optim Analysis software whether in the wizard mode o
141. s window will send an exported file to the designated folder e Close the dialogue box when you are finished by clicking the cross in the top right hand corner You can also opt to export the raw data Raw data is data collected directly from the CCD camera without wavelength device response or background corrections having been applied Raw data Is always stored in the database so that the user can load an old set of data and export the raw file at any time after the acquisition To export raw data e Right click on the spectrum in the list and choose Export Raw Spectrum e Alternatively click the EJ EXPORT button In the dialogue box select a single soectrum to export or click Export All Raw Spectra e Close the dialogue box when you are finished by clicking the cross in the top right hand corner To download the xml file e Click on the blue ID of any of the spectra listed to open an external dialogue box with the options to open or save the file These files will be in an open xml format which contains all raw data calibrated data and a header listing all the settings of the instrument at the time of acquisition Any files accessed via these links will be saved in C Users Downloads P 2 Avacta age ANALYTICAL Avacta Analytical Ltd 2012 IX USER MANAGEMENT Users This tab allows the Administrator logged into the Optim Demo to add users to the database define the permission levels of the user an
142. spectra can be viewed grouped analysed and reports can be written and saved Calibrations A set of mathematical functions that are used to process the raw capture image into a spectrum e g Background subtraction wavelength calibration device response calibration Spectrum A calibrated image from the CCD camera that gives information about the intensity of light at different wavelengths Collection A user defined or default selected group of either spectra or datasets in the Analysis Software that will be carried forward to the next step in the analysis Process Ordinate The property of the data that will be plotted on the y axis of the graph Abscissa The property of the data that will be plotted on the x axis of the graph Primary Analysis Analysis of each corrected spectrum as defined by the selected macro This level of analysis yields a single plot for each cuvette well and for each analysis technique e g An unfolding curve such as fluorescence peak position versus temperature Secondary Analysis Analysis of the unfolding curve primary results for each of the cuvettes wells and analysis techniques to identify transition points e g melting point Tm Tertiary Analysis Multi cuvette analysis Comparing the transition points secondary results in different cuvettes e g Effect of different formulations or consistency between replicates OPTIM 1000 MANL INSTR Monday 06 August 2012
143. splay a large selection of data in a huge variety of different ways using a plethora of functions and user definable methods Data within the system is stored within Studies Each study can contain many runs that can be analysed The runs can be loaded from any Optim 1000 instrument that is running version 1 5 of the Optim Client software A study in the Optim Analysis software is similar to a project in the Optim Client software The two main differences other than the name is that a study can be passed around between users and computers and viewed edited at will QZ TE 7 gt lt Z lt 2 A O Lu ale HANDBOOK A first look To start the Optim Analysis software ensure that the USB dongle Is inserted and double click on the Optim Analysis icon that is on the desktop This will start the software and you will be presented with a splash screen and then the default application screen will be displayed The default view of the software is the wizard mode start screen Toolbar gt Welcome to the Optim 1000 Analyser v1 5 Wizard Seiect the Sudy for your data Load Run for Analysis Choose apea ee No Study Selected _ Load New Run Data d Go to Primary Analysis gt Ayacta ANALYTICAL Wizard start screen P Avacta ogee ANALYTICAL Avacta Analytical Ltd 2012 AOOGAUNVH a m O mA a gt Z gt ins lt w m 7 The
144. t Q LU ale Vat B O1 S FindEdgeLoc InoutData Spectrum to be acted upon RangeMin Minimum value of range RangeMax Maximum value of range Fraction Fraction up the edge for edge location usually 0 lt frac lt 0 5 EdgeName Name of Results File This function gives as the output the estimate of the start of an edge in the InoutData This is roughly the Fraction of the edge height The data are interrogated within the given range only IntegrateArea InoutData Spectrum to be acted upon RangeMin Minimum value of range RangeMax Maximum value of range IntegrationName Name of Results File This function determines the area under the InoutData between the given limits IntensityRatio InNoutData Spectrum to be acted upon NumeratorValue Centre value for integration numerator of ratio DenominatorValue Centre value for integration denominator of ratio FullWidth Width for each integration RatioName Name of Results File P 12 Avacta ee he ANALYTICAL Avacta Analytical Ltd 2012 AOOGAUNVH 4 ais m Q gt Z gt Lice lt n m 0 This function calculates the integrated area under the DataSet for the two regions whose centres are given and with the given Width A ratio of the two areas Numerator Denominator gives the output value BarycentricMean InoutData Spectrum to be acted upon RangeMin Minimum value of range RangeMax Maximum value of range BarycentricNam
145. ta Analysis Engine Beta Development Aya cta Manager Advanced Analysis Centre Report Generator O 20202020222222 O MA ANALYTICAL Primary Secondary Tertiary Method Developer Preview area Level cycler Saved ww methods Function Method list Function generator editor The screen is divided up into six different regions When developing new methods this is the direction of work flow through the system Preview area the data for which a method is being developed is shown here Level cycler this shows the analysis level that you are editing functions for Clicking CYCLE THROUGH LEVEL allows you to change from primary analysis functions to secondary and tertiary analysis functions Function list this shows a list of the available functions for the analysis level that you are looking at Details about these functions is given later Function editor here the parameters relating to a particular function can be edited Method builder here the functions are combined to create an analysis method Saved methods here the methods are saved for use in the software Methods can be saved by clicking STORE METHOD deleted by clicking T and renamed with 4 Saved methods can be loaded into the method developer by double clicking on them in the Saved Methods box Once the method is loaded into the method developer y
146. tc and a dataset P 102 Avacta en ANALYTICAL Avacta Analytical Ltd 2012 3 MeanValue Scatter266 counts nm x10 Well Run Data Folder gt run000140 Data Folder gt run000144 If there are multiple runs contained within the study and secondary analysis has been completed for both runs each run will have a different colour that can be seen through the key at the bottom of the graph Right clicking on the graph will allow error bars to be shown or hidden Tertiary Analysis Tertiary analysis looks at inter well variations in all parameters such as primary analysis results like peak position fluorescence intensity efc secondary analysis results transition temperatures aggregation onset temperatures and other special functions such as Debye plots ee iinne wo E eS OptimAnalysis Analyse Data OptimUtilities I Optim Advanced Data Analysis Engine v1 54 Development Ava cta Manager Advanced Analysis Centre Report Generator ANALYTICAL Prima Seconda Tertiary Method Developer Temperature 19 973 C Ratio350_330 TransTemp Run Time A1 B1 C1 D E1 F1 G1 H1 1 J K1 L1 M1 N1 01 P1 A2 B2 C2 D2 E2 F2 G2 H2 12 J2 K2 L2 M2 N2 O2 P2 Well Run run000172 Collection of Selected Analyses Saved Analysis Collections 1 mg mL Poly IgG in 0 02 MOPS M with 0 M GuHClatpH8 Poly IgG a 1 mg mL Poly IgG in 0 02 MOPS M with 0 2 M GuHCl at pH Poly IgG E 1 mg mL Poly IgG
147. the report When the secondary analysis has been finished clicking GO TO TERTIARY ANALYSIS moves onto the next step of analysis Performing tertiary analysis After completing the secondary analysis clicking ANALYSE RESULTS will perform tertiary analysis This involves looking at the information provided in the formulation file during the configuration of the run in the client software detecting whether there are any clear trends and analysing these If this information hasn t been entered correctly or needs to be reviewed this can be accessed using the sample information editor by choosing View and Edit Sample data trom the Optim Utilities menu at the top of the screen This will be Covered in greater detail in the advanced analysis section of this manual Typical trends that will be analysed are changes in any parameter with pH concentration of sample or additive analysis of replicates to generate averages and standard deviations and Debye plots with sample concentration to determine second virial coefficients The screen will give a graph with results displayed and a key underneath that indicates what the curves and points are and give the result of any analysis P Avacta Se ANALYTICAL Avacta Analytical Ltd 2012 Graph with data and fitted Curves Trans Temp BarycenFluo C Any results Key to the graph Conc Additive 8 Component Additive Data Point ae Fitted Curve Turning Point 46
148. the instrument Hardware installation should be completed before beginning the software Z O lt fa lt o FE a A You should not unpack the instrument until the installation engineer is present An Avacta Analytical qualified engineer should guide you through the site preparation and installation of the Optim 1000 so the following section is for information only Before installing the Optim 1000 in the workplace you need to prepare the site for installation according to the guidelines in this section At the end of the section there are a set of checklists to helo ensure that all the preparation tasks have been addressed Poon lf the Optim Instrument is moved after it has been installed it must be re qualified by an Avacta service engineer before use System Components The Optim 1000 consists of the following items Main Optim 1000 unit External PC with keyboard and mouse Monitor Connector cables Dimensions and weights The dimensions and weights of a typical Optim 1000 system are given below Ensure that the installation site can accommodate the dimensions and weights ceo wan om a wa 85 cm n i 75 kg 165 Optim unit 34 69 cm 28 76 cm 30 Ib Monitor amp Keyboard Standard size Desktop PC pan 43 cm 17 37cm 15 10 kg 22 Ib Avacta Analytical Ltd 2012 OAyacta Page 7 ANALYTICAL 8 m a gt me gt Z Clearances Once in operation th
149. the static light scattering intensity called ScatterMethod and a method to analyse characteristics of the intrinsic fluorescence IntrinsicFluoMethod e Intrinsic Fluorescence o Calculates the integrated intensity of each spectrum in the wavelength range 280 460 nm The result of which is called Fluolntensity o Determines an intensity ratio between the spectral intensity at 350 nm to that at 330 nm called Ratio350_330 o Evaluates the expectation wavelength barycentric mean of the spectra in the range of 280 460 nm The barycentric mean is effectively the centre of mass or a weighted average of the fluorescence emission and is given by A dia fxa da f called BarycenFluo o Determines a peak position and a peak height by fitting a Lorentzian function to the fluorescence initially centred at 340 nm with a width of 20 points and performing five iterations called FluoPeakPos and FluoPeakHeight respectively e Scattering Intensity o Calculates the integrated intensity over the wavelength range 261 271 nm called Scatter266 o Calculates the integrated intensity over the range 468 478 nm called Scatter473 There are also two further methods which are called ScatterMethod_BufferSub and IntrinsicFluoMethod_BufferSub These provide the same functionality as the other two methods but include an additional step where the software will match the provided characteristic information about indiv
150. ts and what they mean refer to later sections in this handbook P Avacta age i ANALYTICAL Avacta Analytical Ltd 2012 XIV THE OPTIM ANALYSIS WIZARD The Optim Analysis wizard is the default method for analysing data in the Optim Analyser It allows a simple few click analysis experience using a selection of intelligent default analysis methods to display the resulfs you want In this section of the handbook you will be guided through the process of loading some data performing each of the analyses in turn and preparing a sample report Loading data into the wizard The first step of any data analysis is to load data into the wizard to analyse There are three available options 1 Load newly acquired spectra into a new study 2 Load newly acquired spectra into an existing study 3 Load an existing study and reanalyse or view the previously analysed data The opening screen of the wizard presents the start of the process Welcome to the Optim 1000 Analyser v1 5 Wizard Select the Study for your data Load Run for Analysis Choose No Study Selected _ a Run WF Load New Run Data QZ LU Y gt i lt Z lt Q O LU T Vath B 1O1 S Go to Primary Analysis gt Name of New Study Kipre 7 Load Saved Study To load data into the system either 1 If you need to create a new study to load your data first enter then name of the study and click on the
151. ue Scatter266 counts nm x10 o oO 3 e 0 05 M Phosphate Citrate at pH 4 6 1 mg ml poly IgG in 0 05 M Phosphate Citrate at pH 4 6 2 mg ml poly IgG in 0 05 M Phosphate Citrate at pH 4 6 3 mg ml poly IgG in 0 05 M Phosphate Citrate at pH 4 6 4 mg ml poly IgG in 0 05 M Phosphate Citrate at pH 4 6 5 mg ml poly IgG in 0 05 M Phosphate Citrate at pH 4 6 Sample Reference 4 Plotting a datatype of Kc RO only available in certain circumstances against sample concentration will disolay a Debye plot Default parameters for solvent refractive indices will be used in this analysis see section in advanced analysis for more information about performing these types of measurements P Avacta aee ANALYTICAL Avacta Analytical Ltd 2012 K c Ry 1 Dax10 Conc poly IgG Component Candidate mg ml There are a large number of potential graphs that can be displayed here Choosing to display the results as a table will display all results for a particular data type and x axis combination In some cases in particular with Debye plots or trend analyses there may be multiple trends occurring in the same dataset For example if a formulation study is being done with concentration gradients of NaCl in both citrate phosphate and phosphate buffered saline There will therefore be two sets of data each of which shows variations in transition temperatures etc with concentration of NaCl QZ LU Y gt i lt Z
152. um was acquired the temperature of the sample at the time the set temperature the laser power measured the time from the start of the experiment the exposure time and the time and date that the soectrum was acquired This information can be represented in the analysis system by a display label and this can be set in the LOAD DATA settings Changing the Display label will change the labels through the analysis software whether it is in method development tables or graphs of results Spectra Parameters to be loaded from Spectra Files Name in File Default Unit Display Label wellRet Well actuallemperature Temperature setpoint emperatur i Set Temperature actualPower Laser Power actualTime Run Time exposurelime Exposure Time timestamp Time Stamp Copy fram Study P 4 Avacta age ANALYTICAL Avacta Analytical Ltd 2012 If you edit these settings you can save them as defaults by clicking SAVE SETTINGS in the GENERAL settings Clicking RESET will reset them to the original saved settings if you have changed then The ANALYSIS settings allow you to change which functions appear in the primary secondary or tertiary analysis sections of the Method Developer Checking the relevant column will make that function available for each analysis level Select Functions Function secondary SubBackgroundRange SsubBackgroundRange Rescale Absolute Sign Integrate Differentiate smooth Heavyside EstimateNoise Si
153. ument should prevent the door from being opened whilst the stage Is hot With correct operation of the instrument the risk to the user Is therefore very low What further No further action is necessary as the risk Is very low action is necessary By whom Wheng P l Avacta age tee ANALYTICAL Avacta Analytical Ltd 2012 6 Lasers AN DANGER Laser Hazard Direct or indirect exposure of the laser beam to the eyes or skin may cause harm Who might be harmed and how What is being done already What further action is necessary By whom Whene User during sample analysis the samples are subject to exposure To one or more laser beams The user may be harmed if their hands fingers intercept this beam In normal operation the shutters in the laser beam will only operate when the sample loading door is closed With the panels in place and the door closed it is highly unlikely for any part of the user to be able to intercept the laser beams With correct operation of the instrument the risk to the user is therefore very low No further action is necessary as the risk Is very low Avacta Analytical Ltd 2012 OAyacta Page 134 ANALYTICAL 7 Relocation Posen Physical Injury Hazard Moving or lifting the instrument and or associated computers may result in injury to the musculoskeletal system Do not attempt to move or lift the instrument without the assistance of others the use
154. ure the spectrum of M1 will be subtracted from that of Al before analysis The soectra of M1 will not be analysed b N1 is the buffer of Fl and H1 with a comparable outcome as above cC O1 Is the buffer for 11 and at each temperature the spectrum of O1 will be subtracted from that of I1 before analysis The spectra of O1 will be analysed Page 44 Avacta B ANALYTICAL Avacta Analytical Ltd 2012 d P1 is the buffer for L Now would be a good time to fill the micro cuvettes with the sample formulations and check them against the list disolayed on your screen The next two sections will describe how to prepare and load the micro cuvette arrays MCAs Preparing the Micro Cuvette Array MCA For making measurements with the Optim 1000 the user can purchase a quantity of Micro Cuvette Array sample holders MCAs Preparation of samples for use in Optim Is not covered in this document For further information about how to handle the sample holders please read the MCA User Guide Warning Take care not to touch the surface of the quartz cuvettes as this could significantly degrade the quality of the data 1 Either use a single channel pipette or pre load the formulations into a row e g A9 P9 of a microtitre plate such as a conical well low volume 384 well plate image Starlabs E1042 3849 P 4 Avacta Se ANALYTICAL Avacta Analytical Ltd 2012 aS lt i i o O NN e w QO mA O lt x M
155. velopment Manager Advanced is Centre Report Generator Files Analysis Li Settings i E AAAA AVACTASERVER lt edirectedF olders daniel lund My Documents Optim Studies a H C Optim Client Database OptimDB Files Capture Default Metho C Users daniel lund AppData Roaming WaveMetrics Igor Pro 6 Packages Preferences C Users daniel lund AppData Roaming WaveMetrics lgor Pro 6 Packages Sample Data FileName Samples Spectra Parameters to be loaded from Spectra Files wellRef Well actualTemperature C Temperature setpointTemperatur C Set Temperature Laser Power Run Time Exposure Time Time Stamp To change where data is loaded from e Select the Load Data option on the left hand side to change the preferences e Under Default File Locations change the Studies and Run data locations to reflect their paths The differences between the three data are P Avacta aaee ANALYTICAL Avacta Analytical Ltd 2012 AOOGAUNVH 4 ais m Q gt Z gt i lt n m 0 Studies collections of runs and analyses that are saved by the Optim analysis software Run data the raw spectra generated by the Optim Client o If this software Is installed on the desktop PC then this path should be C Optim Client Database OptimDB Files Captures o If you are running the software on a separate computer then this path should point to the location to which you are copying data
156. w The user needs fo be trained to not remove the panels Avacta Analytical Ltd 2012 OAyacta Page 131 ANALYTICAL 4 Mains Electricity L DANGER Electrical Shock Hazard The metal outside of the instrument may become live Who might be User Severe electric shock can result from touching live harmed and electrical conductors how What is being All live electrical wires inside the instrument are firmly attached done already to terminals The risk of these becoming detached is low If any were to become detached this would be detected by the power management system inside the instrument or by the incorrect operation of the instrument All panels and framework that are at risk of becoming in contact with a detached live wire are earth bonded The risk to the user is therefore very low What further The user needs to be trained to not remove the panels action is necessary By whom When P 132 Avacta age te ANALYTICAL Avacta Analytical Ltd 2012 5 Hot Plate Pues Physical Injury Hazard The plate where the samples are located may become hot during operation Who might be User The temperature of the heating stage may routinely be harmed and heated to 100 C The user touching this hot plate may how receive burns to their hands fingers What is being The user should allow sufficient time for the stage to cool done already before moving or changing the samples The instr
157. ye Data O O O 0 Analysis Method General Statistics Replicate Statistics Trend Analysis Debye Flot e Set any analysis parameters you require we will discuss these next P 1 Avacta age Ne ANALYTICAL Avacta Analytical Ltd 2012 Analysis Parameters Cpr BEB Bees OHA w names johda nimes DOHA wee e ss ond nimes OHA w Wee e ss OHA w nemes OHA w Wee ess OHA w nemes OHA Wee e s o oHdp niemes et ohia nimesse o Hda Wum ess 1745 3 t0 7376 6 cou 2573 1 counts nm 34506 to 46213 cour _ O Hd te W IaM E s OHS Wee e ss DOHA we W Lee e ss DOHC p name s ohda Wimeme ss OHA nimesse OHA weame ss OHA Wlmeme ss et ACL OHA w Wee s Ord Wee e ss 2670 1109 2 counts nm Mean Std Dew al 3 701 View the result on the graph and match the values shown in the Results box Cycle through the datasets and data types until you have the one that interests you as before data in different runs will be in different colours lt LLI N gt lt O LLI N gt lt Zz 1 lt a a OHA Wee e ss E Ji Cee i i i i bi i di ii il aa r E SRB R EES F ERRE t i vV JO HARRIS AZ h UIM W aayo apydooud 60 0 G a O co TL 0 Hd 1 IOHND Wz UNM W 8914O B1UAdIOUA S O 5 ma 1 O O Olli JO HMw lone Wz uam W epayo apaydeoyd go 0 5 2 Sco Jobe ono neum n uoaa soo i Ept D O_o tonne me s uim n swowmuaoya so o Z GER o yn if 0 Hd W IO
158. yed category in the plot Statistics Well Group by All data is analysed together Duplicate Different duplicates within the collection are analysed separately Combine Grouping spans different runs RUNS No Grouping in different runs are treated as different Replicate Category Reference Number Changes the displayed category in the plot Statistics Sample Reference Well Replicate All All replicates within the collection are Number analysed individually Only replicate number x is analysed Combine Grouping spans different runs RUNS No Grouping in different runs are treated as different Trend Whatever A list of available numeric abscissas Parameter available determined from the sample information provided RUNS No Grouping in different runs are treated as different Match List ABC XYZ List of differentiator sample parameters For example if have trends in pH with two different concentrations of NaCl QZ LU Y gt i lt Z lt Q LU ale Trend Analysis Data Number When multiple results are available for example two transition temperatures for a particular sample this choice determines which to use Fit Type Fits a line to the data in the form y mx c Fits an offset quadratic of the form y y C x Xo Fits a two state thermodynamic model to the data Page 109 Avacta ANALYTICAL Avacta Analytical Ltd 2012 Vath B 1O1 S Deby

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