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Biacore T200 version2 日本語取扱説明書 応用編

Contents

1. CN hm Pe
2. GE D 169 0073 Ye 3 25 1 Intertek 1S 00100 TEL 03 5331 9336 FAX 03 5331 9370 e mail Tech JP ge com
3. ooooe DMSO 1 1 500 RU DMSO DMSO 10 RU
4. Evaqluation Explorer inetics 7 finity Inhulin Backspace Kinetics 7 Affinity Biacore T200 136 5 Toolbar a Kinetics ffinity Kinetics 7 Affinity SelectiGuryves Create 5glect walualinhi mode Single mn Balch modes Pures 1 I H 13 H 3 Znnm lnck S how ohneehntratior series 5 how blarkE Show awerage blarnk s ultiple Hmas dust Inection Start Temperature
5. 60 4 Z 2 a O O
6. Binding to reference Stability baseline a Biacore T200 4 4 1 Biacore 5 A B 2 B 5 A s RU ss
7. OFF TEL 03 5331 9336 ED 9 00 17 30 FAQ Q amp A www gelifesciences co jp gt FAQ Q amp A TEL 03 5331 9336 MicroCoal
8. AbsResp Baseline RelResp Bqseline RelResp Binding level Evaluation Explorer Biacore 1200 180 7 Toolbar Immunogenicity nn ifmatinn Report Point stability vy Response Type Relative Response Report Point stability Show all sample cycles Show al sample cycles Show sample cycles mixed with drug wv Show sample cycles mixed wi ho ru Hegions amp Boundaries Regions amp Boundaries Number of Regions 2 ADAs
9. Table Blanks from other sample series 3 Rejected Cleared Flagged Flog IE Tools gt Hide Rejected Reject Clear Flags Flag
10. DMSO 5 DMSO DMSO DMSO 1x PBS no DMSO 4 6 DMSO 4 DMSO 1x 9600 Ll 100 DMSO 400 ul 10000 ll 6 DMSO 1x 9400 Ll 100 DMSO 600 ul 10000 ll 4 6 DMSO 8 DMSO 49 DMSO 100 200 300 400 500 600 700 6 DMSO 700 600 500 400 300 200 100 700 700 700 700 700 700 700 700 ul Biacore T200 56 3 3 1 Toolbar Run Wizard Menu bar Run Wizard
11. wizard Kinetics Affinity 5 Kinetics Affinitby 2 1 Kinetics Biacore T200 3 Plot QC Biacore T200 1 2 Sensorgram dAll sensargrams RU Sensorgram Plot 800 ua Baseline Capture uaa Baseline Sample gaa Binding level 7nn aa Bindina stability gaa Binding to reference Feport Point Table BE mm Report Point Table Zoom Lack S00 nxiRituxi 400 ef nxid Ritux amp Startup 300
12. 1 Median Filtering Uese median filtering Y axis X qxis Settings Median filtering Plot definition Report point bindina Response type Fielative Fiesponse kMedian Filtering settings 0 median hkermg Zoom Lock Curves Fc 2 1 corr Y axis Tatal points 128 Above range 45 Upper limit 10 In range 78 Lower limit 5 axis Below range 2 Window width 10 Before filtering After filtering ty a 2 Ye i 3 Eg g Biacore 1200 22 1 hedian Filtering setiings HE pper Im LnwEr lirnit Window width Y Upperlimit Lower limit RU X Window width
13. OO gt GO6 96 9 9969 O00000e O00000e OO O00 OOOO Biacore T200 1 3 7 Menu gt Export Positions Menu Sample Position Import 1 4 8
14. 4 2 Menu Export Positions Menu Sample Position Import Biacore T200 4 79 4 3 8 Control Sqmple R1A1 R1A12 12
15. NSB Reducer BR 1006 91 Merged Injection 1 mg mL HBS EP HBS EP 150 mM NaqCI 0 05 Surfqctant P20
16. Exclude Result Plot Plot Plot a Flot i Easelirne Sample 1 Bindinn lewel binding Binding stability stability i Bindin to references stabilty gn Larru nBl co_binding i Controls hindin binding gia Controls stability stability Plot Biacore T200 1 13 Plot Binding level Plot
17. Menu Export Positions Menu Sample Position Import Biacore T200 62 3 3 5 8 Control Sqmple R1A1 R1A12 12 060327 Thermo Rack Positions Reagent Rack 2 Type Sample 1 3 E 14 Negative control Control sample 4 i 148 Negative control Control sarnple i 148 Negative control Control sample ww 2 E 1
18. AbsResp Baseline RelResp Bqseline RelResp Binding level Evaluation Explorer 5 133 Kp kgkg BiacoreT200 Toolbar nd Kinetics ffinity Kinetics 7 Affinity Selert Curyes Create Gelect ewaluation mode Single modes Batch modes Lurwgs Contact Time Diss Time 5 5 1 II5 H 13 H S ho oheehntratio series 5 ho blarkE Show awerage lankls Select Evaluation mode Single mode Next Biacore 1200
19. Toolbar Run Method Menu bar Run gt Method Te 0pen7New Methnd Laok jn Methods And Templates Name 1Biacore Methods Show importqble wizard templates 0penNew Method Lookin 100322 Name Tupe kodified Immunaqenicitu Screening Immunanenicity Screening 722 2 1 Show importable wizard templates Open Method Builder Moin Overview BiacoreT200 Biacore 1200 Cycle Types Method Builder Main Cycle types Description of selected cycle type Screening General Settings Conditioning Assay Steps CucleTupes 2 Wariable Settings Commands Report Points
20. Ctrl Breakl _ Biacore T200 Evaluation Softwqre Biacore 1200 10 1 1 2 Evaluatton CIBiacoreT200 Evaluation Software 101207Parp15 fr screen_box2_lah60 hlr a He H 3 rd Sensorgram qmPFlat lt a Bar Chart Aa Kinetics 1 AFFinity gt Concentration Analysis Thermadynamics Immunogenicity gt A Screening v All sensnrgrams dL Curve Name Fc 2 1 Assay Sten Purpose lt Dwerlay gt Cucle lt Hverlawy Sensorgram all sensorgrams RU Sensor gram Po 8000 Zoom Lock uaa Baseline 5ample Bindina level gaa Binding stability ga Binding to reference aa Earry over Controls binding aa Controls stability Hepart Point Table mm Report Point Table i a Solvent Correction
21. 1 0 5 TFA 0 1 ZipTip Milipore 50 mM NoOH
22. Biacore 1200 7 187 7 9 Menu Export Positions Menu Sample Position Import Immunogenicity lsotyping Prepare Run Protocol 10 7 D Prepare Run Protocol e ake sure the carrect sensor chip IS docked e ake sure all sarmples amp readents are Inaded in the rack and micrahlate according tn the Rack Positions setup ylals should be sealed with rubber caps and micrahlate with adhesive foil e Flace the huffer si nn the left hand tray and insert the canrrect tubing s see below Notel Standhy after run will use buffer A e ake sure there is fresh water in the water battle
23. Calibration Curve Gee 1 lt Lurwgi Ctrl Calibration Tahle Calibration urye Lurye Hverlaw gt RU Biotin 1BQ0 1380 5 14nn 1298 5 1200 1000 SOD Relative Respornse BOD 400 Parameters 20D lt Dverlay gt 20 2 40 BI gD 100 120 Concentration nd Use calibration trends Calibration Table Conc CalcConc
24. Settings Curve Report Point 2 enhance_level 2 stability Show sample cycles mixed with Regions amp Boundaries Number of Regions Boundqry Region Name High low 7 5 Finish Biacore 1200 7 181 Biacore T200 Evaluation Software C ABia Users T100 ver2_demo data immunogenicity Ex 4 Confirmation bme Confirmation Fle View Evaluation Evaluation Explorer 1 x Remove Edk Sensorgram 9 All sensararams Plot ga Baseline Sample a Binding stability gi Controls stability Hepart Point Table mm Hepart Poi
25. Keyword table Tools Keyword Table Edit Chip Information 1 i 5 1 fifi 11 RNN NN 11 ee me ee ss me se ss se ss EE Biacore T200 7 8 Menubor Toolbar Sensorgram JA sensorgrams Plot ga Baseline Sample Binding stability ga Enhancement Peport Point Table Evaluation Explorer Work area Menubar Toolbar Evaluation Explorer ororam Pot Baseline Binding level Binding stability Binding to reference Work area mM Report Point Table 7 179 Curve Name Fc 1 44 Assay Step Purpose lt Dverlay gt Cycle lt Dverlay gt RU Sensorgram 75000 9 Zaam Lock ga Controls enhancement 7Unnn Se n SO rg ra m WI n d OW gi Controls stability ESOO0 Ul I Ei 55nnn Cortrol Samhle 1 Saimple amp Startup Sn 8nn 1000 1200 1400 1BQQ i s
26. Thumbnails Status Biacore T200 2 Kinetics Affinity 51 Tools Hide Rejected Reject Clear Flags Flog 2 2 Results Summary KOD Plot Table Affinity Screen Fit Settings Tools gt Clear Flags Sample 1 Ligand parp15 Status IE Cleared gt Tools r Thumbnails Results Summary Response Concentration Plot mi Table Calumns Sensorgram KD Plot Zoom lock Disnlay Blanks Display Blank Subtracted Samples Included Curyes Result and Parameters Control Rmax RU 100 Da Rmax Adjustment Positiy 8 5Ze 05 5 Z1e 05 b amp 15e 05 4 8b6e 05 3 51e 05 9 88e 05 9 24e 05 4 97e 05 1 21e 04 KD Plot Plot
27. Eject Rack Roack tray port OK Eject Rack Truy Rack Positions Next es Immabilization pH Scouting Prepare Run Protocol Prepare Run Protocgl ak sure the correct sensor chip IS docked akg sure sarmples Meadents are loaded in the rack and rmicroplate according to the Rack Fnsitinns setup Pials shnuld be sealed with rubber caps and microplate with adhasiwg foil Flace the hufferrsl on the left hand tray and insert the correct tubingls see below ntel Stahndby gr Fuh will use butfer a kakg sure there is fresh water in the water bottle on the Fight hand tray If necessary Bmpty the waste bottle betore start of the run Estimated ru tirme 18 min lewcludinn conditional statermernts temperature charnges and starndby Ho Estimated uffer cornsumptior plus SS mday for standhy after run start
28. Abort Remaining Biacore T200 50 2 Kinetics Affinity Affinity Screen Fit Settings Taols Clear Flags Sample 1 Liqand parp15 Status IE Cleared gt Tools r Thumbnails Riesults Summary Response Concentration Plot View vy Arrange By v Sensorgram Zoom lock a lparp15 TC Display Blanks 8 Display Blank Subtracted Samples Included Curyes Result and Parameters Report Parameters Rmax RU offset RU Control Rmax RU 100 Da Rmax Adjustment Positive Control Adjustment Fitting Fu N Thumbnails Thumbnails 4 Rejected Cleared Flagged Flag Accepted E
29. Deposition solution volume Ll Number of repetitions 1 10 Biacore 1200 146 6 Description of selected cucle tpe anditianin of the surface is performed by injecting a regeneration solution General Settings This solution is the typically the same as the solution used to recover the material captured nn the sensor surface Assay Steps Tupical recowery solutions that are MS compatible contain mild oranic acids 0 2 1 way tnifluoro acetic acid TFA formic acid or acetic acid Cycle Types Variahle Settings Commands Report Points ce rg Rogereion Rgeneration solution 0 5 TFA Set property as variable ime 3H 8 i Heqeneralion solution klethod Variahles Flow rate Flow path Fredip Flow rale plimin Setup Bun Riegeneration 1 Mymin Contact time s High yiscosity solution Estra wash ater inlection with LN Stabilization period p s
30. OQCratio 0 2 lt 0 2 Biacore 1200 4 117 Cycleg C Lnand hilutinn FlInw Initial ratE QC ratio Temp OR HG fartnr Wulnmnin nreI RU s1 prel LL 1 1 Inrlure wl 1II 1 93 We Fr 4 3 1HHuajmml Prntein A pHS 0 mouse I G 1 H i 1 391 eq 15HHHH ww 12 5 ES 13 1 1 Fr 4 3 1HHuajmml Prntein A hH5LH mouse I GTx1J1 ni 1 nd q 1S5HIIHH 14 1 15 1nn 26 5 Include ie 5ampls mouse m1 Curwe Fc 4 3 jie Show blank subtracted datq Advanced settings dyainrB settings pply settings to Lut range All sample series Sinnle sampl
31. 5 2 Menu Export Positions Menu Saqmple Position Import Biacore 1200 128 5 5 3 8 Control Sample R1A1 R1A12 12 OOOOOOOOOOOOI
32. KineticsyAffinity System Preparations Frime before run Mnrmalize detector Temperature settings hasis emperature CE Sample compartment temperature LE me Les Le Prime Normalize Anqlysis temperature 25 C Sample compartment temperature 25 C Cycle Run List Tee Lnnrentratinn Analysis Cycle run list hssay step name Sample 1 5nlutinn sample 1 Conc ng ml sample 1 Dilution m tartup iu tartup Car 3 tartup im 4 Calihratian Bigtin 1nn 5 Calihratian Bigtin 40 6 Calbratian Biatin 15 Calibratinn Biatin 5 8 Calbratian Biatin 2 55 9 Calbratian Biakin 1 2 10 Cnrtran 5ample Cnrrtrnl 1 33 3 11 Control Sample Control 2 11 1 12 5ample 501 2436 01 1 13 Sample 5n1 2436 1 1 Next Biacore 1200 78 4 es Concentration hnalysis Rack Positions Fieagent Rack 2 dE well Microplate 1 1 1 L 1 1 1 Content 11
33. Biacore T200 Empty Recovered sample min capac 19 0 5 TFA 521 0 5 TFA 427 5Hm NH4HCO3 ity ZOul 6 149 6 1 8 Control Sqmple R1A1 R1A12 12 060327 Thermo Rack Positions Reagent Rack 2 Type Sample 1 3 E 14 Negative control Control sample 4 i 148 Negative control Control sarnple i 148 Negative control Control sample ww 2 E 148 Negative control Control sample i 148 Negative control Control sample E 148 Negative control Control sample s 22 148 Positive control Control sample F i 148 Positive control Control sample 96 Deep Well Microplate 1 148 Pasitive control Control sarnnle 148 Positive control Control sample 148 Positive control Control sample 1 14
34. Kinetics Affinity Biacore T200 1 2 Evaqluation Add Solvent correction gnlwent nrrertinr Zoom lnck im BI ie 0 5 Eslrannlalin Hanng Report PFnint Hanng 1500 1IHH 5I OD 1 T1500 None Etrapolate le Show rart hiht rah E RespnrnisE Ret OK Evaluation Explorer Solvent Correction Curve Nome corr Fc2 1 corr Biacore 1200 3
35. Table Lae Biacore T200 14 1 Color By 4 Lncle lt erlans jie Fwe wrla Sart Curle Twhe Table Cnlumns we ssay Step Molecular Weight djustment ssay FE Furpose Temp BUFer 5 Ti Negatiwe 5ample 1 1 TO Ligand TIC 7 Tb wy 1n 8 4 T_ 11 i a 1 Show Sensorgram Y 0 Table Show Sensorgrame 5 Exclude Cycle
36. CFCA 100 ul min d R _ f KM K_ Conc7 1 9R SU 5 5 1 amp 100 a 5 ul min Mw Do k CFCA 150 kDa 5000 RU Mw km Conc d R dt 2 5 100 ul min km D 2
37. Browse Open es nnrentratinn nalysis InjErtinrm Sequence Liatechinh Fhip Captue A TPLE amplg Enhancamert REGEMNERATION 1 Heqeneralian 1 Biacore T200 Detectlon Flow path Chip Chip type Capture Sample Enhancement 2 Regenerdtion 1 or 2 Next gt nnrentratinn tnalysis Setuh Lnnditinnin Hun canditianing crcla Lnntact himg Mumher of inections Startup Hun startu
38. Low Medium Table Ranking Low Lowest Points Medium Low Medium Medium Vaqlue Name value Name Edit New Biacore 1200 1 25 Cut off Mode Cut off 3SD Cut off Edit Settings Cut off
39. http www biacore com diffusion_calculator FFINT Diffusion Coefficient Calculator Converter This on line tnnl Is desI hd tn helh nu raculate diffusinn coefficients tor sein Calibration Frees nrghtratinh Aha vss 55ays ti accessible only via a walid product ky associated with the abprophriate hes of Biacore SnftwyaFg eed to be goed in hd heave WT Produet er registered pF aceonht detlas Fo Ho hot hawe 3 Biacote WE aCCO UTE et ol gh ete FOFiGOT FHSSIIDF LD L IF User ID Password LOGIN Biacore T200 94 4 Diffusion Coefficient Calculator Converter ThIS on line tnnl 5 desidhned to help vou calculate diffusion coefficients for use ih Calibration Frees Coherentration r naysis 558ays IE aresSsible nhly a a val roduct key associated with the ahhropriate thes of Hiacore nftyarB Calculate diffusinn cnefficient nt 20 C l l l l Niolecular weight IIHH a l l Frictional fio wm Choonse molecular shabe hular TT l Enter walue l WISCnSIW Telitrve Lse standard walue Hi 1 IT W ET a OC Enter walu
40. Following Blank Molecular Weight Adjustment 100 X RU Do Keyword Table Y 100 Xx RU Da Result Plot 1n0 RUDa Result Plot cn cn ms Lm Relative Respornse binding Adjusted oe In 2 4 BI gD 100 Rieport Point amp diustment RankingACut off Curve Fitting Blank Subtraction NN holecular weight Adjustment 4 Capture Adjustment djustment For Controls Median Filtering Biacore T200 Curve Name Fc Corr Assay Step Purpose lt Dwerlayy lt Lcle lt Hverlay gt rj 120 140 Blank Subtraction Blank sample negative Subtraction Tpe Nearest Blank hiolecular eight Adjustment Zoom Lock Control sample Sample
41. _ Blank Subtraction Blank Subtraction Use Blank Subtraction Blank sqmple name sample Conc 0 Blank Suhtrartinn Settings se Blank Subtraction Blank sample name ubtraction sethings earest Hark kJ Mgarmst Harnk werane Nearest Blarnks Frecedinn Blark Fllnnn Bark Biacore 1200 18 1 Subtraction settings Nearest Blank Average Nearest Blanks 2 Preceding Blank
42. Tools gt Hide Rejected Reject Clear Flaqgs Flag 2 2 Biacore T200 2 Kinetics Affinity 35 2 2 Rmax lt KinEtir SErEen FE 5etHings Clgar Flags ample 2994 Removwe RaTIHE5 ThumhnalE Fasuls NT mas WIBWU rtrange By Print 2 2 dd ef oxid Hilug Tools Annotations Naname GustomiAnnotation HH nnntahinn dd Lammer sa hnntahinn Template Savwe drnnotations As Template Add Annotation Annotation Add Comment
43. 3 Next Biacore 1200 160 7 Immumngenicity SEreEning InjectionParameters 5ample antact Hm gl Flo rates hg Mrmnl Eid solutior 1ghU mM HLL 1 PE gutralzahin snlutinrr Th THI5 HE Pa Enhancamahnt 5nlutinm 5heen arti rabbit lig Corntact kime ls Flo rate 5 mml First renerlerahinrn 5nlulinn 1 5 MM BLA 5H mk HLI High wiscosity solution Corntact time lanwrate lmnl Second reqerhneration Golutior 1U mk NaHH High wiscosity solution Contact time 10 lawurate lrmim 5Lahlllgatinh perind nm Eshra wash fer imlectinn with DAD isnnrnn HH 2 2 Next ImmmumngEnmirity Screening SanmnlEs Sample table Sample id Serum 3 samples 5 om Sample 0 5 x100 drug SET 2 erum 3 erum 1 Serum 2 Serum 3 sample 5 poiml Serum 1 Fiurn order Cs ehtered Random Cut off
44. guanidine HCI 10C Cycle Run List Next Biacore 1200 176 7 Immunogenicity Confirmatinn Rack Positinns 3 KO KD Fi 1 67 Neg control Control sample KD KD 57 Neg control Control sample B 57 Neg control Control sample WS 657 Neg control Cnntral sample XD i 57 Neg control Control sample 67 Pas cantralt1 aim Carntral sample OL OS 67 Pas cantralt1 aim Control sample XD 67 Pos cantralt1 aim Control sample E 67 Pos cantralt1 aim Carntral sample dB yell Microplate 67 Pos controlt1 pgirmly Control sample 1 Empty Mix min capacity 79ul Control samnle Mix 3 Empty Mix min capacity 79ul Control samnle Mix QO Empty min Capacity 79pl Control samnle Mix 11 6 Empty Mix min capacity 79pl Control sample Mix 10 8 Empty Mix min capacity 79pl Control sample Mix 5 Empty Mix min capacity 79ul Control sample Mix Empty ma min capacity 79ul Control samnle Mix 9 8 O Empty Mix min capacity 79pl Control sample Mix 7 Empty Mix min capacity 79ul Control sample Mix
45. Merged Inject Sample Acidification solution Neutralization solution Next Immumngenicity lsntyning Samples Sample table arnnl id Next Biacore 1200 7 185 Inmmmunngenicity lsntyning lsntyhini Reagents lsnlunin reagent ahle anti Ig anti Iga Isotyping reagent i Next Immunogenicity lsotyping Cycle Run List Next serum_NSBEred serum_NSEred serum_NSBred AD nl 2 3 4 nA5 nb 7 ats BD B1 B2 B3 4 B5 Be B BG CH anti_IgM anti_IgN anti_IgN anti_IgN anti_IgM anti_IgN anti_IgM anti_IgN anti_IgN anti_IgM anti_IgM anti_IgM anti_IgN anti_IgM anti_IgM anti_IgN anti_IgN anti_IgN anti_IgM anti_IgM anti_IgN anti_IgN anti_IgM anti IqG anti IqG anti IqG anti IqG anti_ IqG anti_InG anti IqG anti_IqG anti IqG anti IgG anti_InG anti IgG anti IqG anti IqG anti IqG anti_
46. Concentration Analysis Create Control Sqmples Goncentration nalysis Create Calibralinn Control Samples Gamples Control Sample Table Control Sample Plot ngitnl Conc Response Calc Conc 40 ng mb ng ml 10 Cantral 1 dd 6 fo 17 Control 1 33 3 33 15 24 Cantral 1 33 3 6 34 78 31 Control 1 33 3 32 59 44 Cantral 1 33 3 34 n1 51 Control 1 Awvg 100 6 Cycle Sample Id 11 Contral 2 6 6 100 1 18 Cantral 2 6 101 2 25 Control 2 6 103 8 32 Cnntral 2 102 3 45 Control 2 6 6 6 10n2 3 52 Cantral 2 Calcuated ConcerWratinn 102 1 4 10 Tools Custom Report Points Wnstom REpnrt Pnints Custom Fieport Fninls Add Biacore T200 90 4 AHH Custnm Rennrt Pnint Hghnart point stahiltg wry Fnsihinn Ihe report point sBCnInHs 5ample 1 injection stop Falculate resnnnse relatiwe to report point Cucles Apply Tn Selected Assay step purpose Gelected hssay step puUrpose tartup Capply Tn Selected Cucles Calhrainn Control sarmple 5ample ld
47. blank Run order Next Biacore T200 7 161 Immunogenicity Screening Control amples 4 Lnrlrnl sarmple defirition Fun cnhlrnl samples Repeat control samplels evern 1 sample cucles Lnnlrnl samples Control sarnnle id gn cnntrnl HH Iml Pns control 1 I jml Run control sqmples Run control sample s every 10 20 Next gt immunogenicity creening System Preparations Erime before run Normalze detector Temperature settings halusis emperature Sample compartment temperature EC Analysis temperature Sample compartment temperature 10C 10C
48. Result Plot Flot Settings Plot name A Etting TS LT Hepot Part Varahle Response Tupe Helalrwe Hesponse 1 Cares Lurwe ame Fc 2 1 cnrr Plot name Y Axis Axis Curves Next Result Plot Biacore 1200 1 17 Yalue Y Yalue Sample Result Plnt Curve Name Fc 2 1 corr lt Assay Step Furpose lt Hyerlaw gt lt Lucle lt Hverlay gt rj Result Plot Zoom Lock 5 1 2 negative 5 1 1 negative 7 49 8 STOSDZ 8 0 1 T 5 9 0 2 T_6 12 3 n Aa 2 13 0 9 A 3 14 1 7 A 4 16 0 2 Aa 6 17 0 1 Aa 7 18 1 3 negative 19 1 3 negative 2 52 3 STOSDZ 21 0 7 A 8 ZZ 12 3 9 23 39 2 10 24 0 6 B_1 25 Dn 1 B_2 26 3 n B_3 Point Adiustment Hanking Cutoff Curve fitting 27 1 4 B 4 28 1 8 B 5 30 1 0 B_7 31 D b5 negative Capture Adjustment 32 0 6 negative ee 33 52 7 STO802 1 36 3 3 B10 Report Point Adjustment
49. Save qs Methods and Templqates Bia Users Don t Save Biacore T200 1 9 irmmohilzation of antibody blr Immabilzation of Prteina blr manual blr re re Thermodynamics_antibody ys antigen e Resut fle bn Save in File name Save Standby flow Biacore T200 Evaluation Software 1 5 Run gt Stop Run Hiacore Tzhn This will stop the run orn Stop Run Runistopped Finishin current cucle please wa bort cncle hn Ct Erek
50. Table Rrmax Cyele lt Ovedlag gt oek Calar By LCut ol Znam Lack Tahle Calumns Edit Annotations oy Tools gt Edit Annotations Naname Custnnm Amnntatinnn HH nnntahinn H nmment sa hnntahinh Template Gavwe dannotations s Template Add Annotation Annotation Add Comment Annotation Biacore T200 1 27 Managa Custnm Annntatinni Bulk response dd natation High UK H nmmenlt hmnmalnus hindin Ho saturation Delete High Rmax se hhntalinh Tamnlats SaveArnnotations A amp s Template Carcel Annotation
51. k k Toolbar Thermodynamies Biacore T200 5 137 Thermodynamics Select Kinetic Data Create Impart Kinetic and amp ffinity Evaluations Ligand Sample antigen Model 1 1 kinelics stead state alfinity Ewaluation Temperature 2 Description ka kd ls KD MM antigen 9 9 1 1 Binding 1 227E b5 5571E 4 4538E 10 antigen 15 15 1 1 Binding 1 1U5E B 342EE 4 8 534E 10 i antigen 23 23 T 1 Binding 1 383E 5 2 0358E 3 1 473E 3 antigen 30 3H 1 1 Binding 1 901E 6 4559E 3 2 39E 9 i antigen 37 37 1 1 Binding 2 258E 5 544E 3 4270E 9 Import Thermodynamics Check All Next Thermndynamics Dveryiew Ereate Thermndunamic yerylew Tammpneraturg a Desoipion ME kd 1 9 1 2 arntigen 1B 15 1 1I5E 5 rhneh 23 eg 1 38 5 nhneh dL dl 1 901E b areh 3F 7 SSE b 2 Temperature 15 3 15 2 5
52. DMSO _ UV spectrometry DMSO pH DMSO pH Biacore T200 54 3 SPR RI 100 RU
53. Commands Sample1 Settings for Sqmple1 Buffer Type High performance contact time s 36 Dissociation time s 5 Flow rate umin 30 ul min Flow path Both Sample solution Buffer Commands Regeneration1 Regeneration solution contqct time s Flow rate ul min Flow path Both Biacore 1200 102 4 Ts Method Builder Main ucle tupes Descrintion of selected cucle type Le lm Assay Steps cceType _Variable Setings Vericaton Commands Report Points Ines a Riemowe Method Variables Ewaluation Variables Set property as variable fample solutior ls wariable Sample 1 Sample solution Regeneration 1
54. Biacore T200 Sensor Chip CM5 Merged Inject Fc3 4 Fc4 ADAs Fc3 150 kDa 7 000 RU 15 000 RU HBS EP 7 155 Merged Inject
55. Number of injections Temperatures Analysis temperatures Repeat after Sample cycles 25 5 Sample compartment temperatures Next eThermodynamics ample Contact hme Injection Parameterns Flnwu rate plermirnl Dissociation time s Era wwash after iniection with vi Hgeneheralinn Solulion High wiscasity solutian Contact time sa Flow rate plemin OStabilieation par Sample contact time 120s Flow rate 30 ul min Dissociation time 120 s Extra wosh after injection with Regeneration Solution Biacore T200 126 5 High viscosity sol
56. Regeneration solution contact time s Flow rate ulMmin Flow path 1 2 3 4 Setup Run hethod Builder Detertion Detection Flow path Flow path 1 2 3 4 Next Biacore T200 Methn Builder Cycle run list Cycle Rssay step name 5urFarg conditioning SurFace conditioning 5urfarg conditioning Inject and Recower Next Tes Methnd Huilder ystem Prenaratinins Frime before run Mnrmalze detector 5 Prime Normqlize Temperdture settings Anqlysis temperature 25 C Sample compartment temperature 25 C Next Biacore 1200 ethod Builder Rack Pnsitinns OC ee OO OO OOOO SooCOO OO OO UO OOO O00 38300 ee eee te eco90 ul
57. AG AH AS 2 2 AH A AS 1 2 1 so EC 29 C 45 C 37C O 45C kJ mol 9
58. Menu Sample Position Import Biacore 1200 7 163 Immunogenicity Screening Prepare Run Prntncnl Tahoma 10 BH U Prepare Ruh Frotocol ak sure the correct sensor chip 1s docked akg sure sarmples eadehts are loaded in the rack and microplate according to the Rack Positinons setup ials should be sealed with rubber caps and microplate with adhgsiwg foil Place the butferls on the left hand tray and insert the correct thing s1 see below ntel Standhy after ruh wl use huffgr A hdake sure there is ffash water in the water hnttle nn the right hand tray If necessary mphty the waste bottle betore start of the run Estirmated run time bh Hmn enclading conditiornal staterments temperature charges ard starndby Ho Estimated butfer cornsumptior 2 Funninn bffer J atleast FUO ml plus 5H mydan for stahdhn after ruh Start Save qs M
59. txt Tes Kinetics Affinity ystem Preparations Prime before run Narmalze detector Temperature settings halusis lemperature LC Sample compartrment temperature FI Prime Normalize Temperdture settings Anqlysis temperature 25 6 Sample compartment temperature 25C Cycle Run List 1 startup buffer 2 Startup buffer 3 Startup buffer 4 Solwent correction 5 Control Sample CB 5n 5 sample Sulfanilamide Sample Sulfanilamide 8 Sample Sulfanilamide H IH79 9 5arnlg 5ulFanilamidg D157 10 Sample Sulfanilamide H 3131 111 Sample Sulfanilamide Hz 12 Sample Sulfanilamide 1 25 13 Sample Sulfanilamide 2 14 Sample Sulfanilamide 15 Sample 5ulFanilamide 1n 15 Sample Sulfanilamide Hg Biacore T200 3 61 Next Tee Kinetics hffinity Rack Pnsitinns 148 5ulFanilamide Sample 148i5ulfanillamide Sample 148 sulfanilamide Sample 148 sulfarilamide Sample 148
60. 0 FAX 03 5331 9370 e mail Tech JP ge com Q amp A Web TEL 03 5331 9315 FAX 03 5331 9349 TEL O03 5331 9330 FAX 03 5331 9324 WWW OelifesclenceS CO D e mail Web 2013 GE
61. Save Annotation As Template Annotation OK Annotation Table Annotations Annotation Comment Table Result Plot Curve Name Fc 2 1 corr 4 Assay Step Purpose lt Dwverlay gt Cucle lt Hyerlaw gt Result Plot Tools Sample Annot Bulk response Annot Anomalous binding STOSDZ Zoom Lock 94 6 Above Cut ofF 16 2 Above Cut off BE 8 0 6171 Below Cut off BE 9 3 786 Above Cut off B_1n 29 19 Abowe Cut off C 1 H 3 849 Above Cut off C 2 5 566 Abave Cut aff C 3 8 1 514 Above Cut off C 4 Cartral sample 3 669 Ahnwe Cut ofF C 5 Sample 10 51 Above Cut aff C 7 3 228 Belaw Cut off negative amp 1 62 Below Cut off negative 99 19 Abowe Cut off STO8n2 26 19 Aboye Cut off cs 0 5615 Below Cut off c9 3 58 Above Cut off C_10 13
62. OK Eject Rack Tray Rqck Positions Next es Thermodynamics Prepare Run Protocaol Prepare Run Protocol akg sure the correct sensor chip I5 docked akg sure all sarmhlas amp readqents are lnaded in the rack and microplate according to the Rack Positions setup Pials should be sealed with rubber caps and microplate with adhmsiwe foil Flace the butfferts on the lett hand tray and insert the correct tubingls see below intel Standby after Fuh wl Use huffgr a Make surem there is ffesh water in the water hnttla nh the right hand tray lf necessary mhtythe waste hnttle before start of the run Eshmaled ru mg 12h 13min excluding cornditional statements temperature channes ad standh flo Estimated buffer cornsumptior Ruhning huffer 0 last ND ml plus 5 mlHay for standbsy after ruh start Save qs Methods and Templqates Bia Users
63. Repeat after _ Sample cycles Next gt Kineticsyffinity Injertinnm Parameters 5ample Contact hme ls Flow rate Imn Dissociation time y Estra wiash aftr inection with Stabilization period s Sample contact time 120 s Flow rate 1 30 ul min Dissociation time 120 s Extra wosh after injection wrth Stabilization period 0s Next Biacore T200 3 59 Tes KineticsyAffinity Samnles Samples Concentration Concentration 5arnnle id 5ulFanilamide 5ulFanillamidg 5ulFanillamide 5ulFanilamide 5ulFanilamidg 5ulFanillamide 5ulFanilamide 5ulFanilamidg 5ulFanillamide 5ulFanilamide 5ulFanilamidg znsulFamidg znsulFamidg znsulfamidg znsulFamidg znsulFamidg znsulfamidg znsulFamidg znsulFamidg znsulfamidg Hun n
64. Cycles OK Gustom Report Points Custom Hannrt Foints MM Posion Assaysteppurpose stahilitw a sECnrids after stop oF alihratinn injgrtinn sample 1 Sampls injection Cantral Sample Coalibration Settings Report Point lhrahinn Lure Sethngs Flow Cel TFc 1 Hehnarl Fan baseline Use awerage calibration ur stabilit stahilhug Biacore 1200 4 2 CFCA Calibration Free Concentration Analysis CFCA Calibration Free Concentration Analysis
65. Don t Save Save Results From Run hs Save in 2 My Hecent Documenls Desktop Mu Documents 9 Mu Computer Mu Netwark ED T10manual immblization of antibody blr Immabilization of Prteina blr manual blr pHscouting blr regeneration check blr regqeneration check_wizard hlr surFace parFarmance_wizard blr File name Thermodunamics_antibody ys antigen MM Save as type FesukfleFbl Biacore T200 130 5 Save in File name Save Empernature djustment Waiting Fnr stable temperature Tn obtain reliable results wou should wait until Fhe bemperature js stahlg et temperature 9 Current temperature Temperature sFahle Unstable Standby flow Biacore T200 Evaluation Software
66. Cycle Run List Biacore T200 6 1 es Binding Analysis Cycle run list cycle Assay step name sample 5oluton Cnrditinnin 2 startup buffer 3 startup buffer 4 Startup buffer 5 Control sample Control Ln i mi Dm mn FP a a Ln wn i Lm le Sample 11 7 Control sample Control Next Te Binding Analysis Rack Positions Se ren 4 118 Control Control sample CD 118 Control Control sample 118 Cnntral Control sample ee 118 Contral Cnntral sample XD XD 118 Control Control sample 118 Cnntral Control sample 1 XO CO 118 Control Control sample 118 1 Sample E 118 1n Sample 96 Well Microplate 118 11 Sample 118 12 Sample B 118 13 Sample ee a OQ i 118 15 Sample 10 3 118 15 Sample OR 5 ee 118 18 Sample 118 19 Sample 118 2 Sample 118 Zz0 Sample 118 21 Sample 118 22 Sample 118 23 Sample 118 24 Sample 118 25 Sample 118 26 Sample 118 27 Sample 118 28 Sample 118 29 Sample wv cgack New gt ges ul
67. Annotation Biacore 1200 36 2 Kinetics Affinity Manage Custom Annntatinn Bulk respanse dd Annotation High IK dd Comment Anomalous binding No saturation High Hmax se Annotation Template Save Annotations A amp s Template Annotation Save Annotation As Template Annotation OK Annotation Table Annotations Annotation Comment Results Summary Table Annotation Thumt ails Fiesults Summary 1 Table Co On Off Rate Map Annot Bulk response High Annot Anomal
68. Biacore T200 Evaluation Softwqre Biacore 1200 3 65 3 7 binding stobility co binding stability co_binding co_stability 2 Carry over Control Regeneration 3 8 Wizqrd Binding analysis Kinetics Affini
69. 67 3 9 Exclude point Exclude ruryg Show Sensorgrams Srale nnw Graph Eh CUrves Exclude point BHH BOO 400 200 0 200 4O00 BOO gO00 1000 1200 Response Ref Biacore T200 68 3 3 10 Solvent correction Table Include 3 Fc 43 15 BHH BOOD 400 200 0D 200 dO EOD BU 1000 1200 Response Ref RU Biacore T200 3
70. enhance_level 2 stability Regions amp Boundaries Number of Regions Boundqry Region Name Finish 7 5 Adjustment for Controls Biacore T200 170 7 hdjustment fnr cnnitrnls Flat definition Hepart
71. CV CV 4 7 4 8 Biacore T200 86 4 Concentration Anqlysis Create Samples Concentratinn Analysis Create Calibration Control Samples Samples Sample Table Sample Flat sample 1d a 12 501 2436 01 13 501 2436 01 14 501 2436 01 15 501 24365 01 wg 16 501 24365 02 19 501 2436 02 wg 33 53 20 501 2437 01 6 18 8 21 501 2437 01 Awg 18 6 22 501 24383 01 23 01 23 501 2433 01 Awvg 26 501 2439 01 27 501 2439 01 20 40 BO gD 100 120 Measured Concentration ngitnl Sample Table Dil Fact Calc Conc CV CV 4 7
72. NaCl Surfactant P20 DMSO Biacore T200 6 1 Toolbar Run Method Menu bar Run gt Method OpenMew Methnd Lookin Methods And Templates Name kModified Biacore Methods Biacore Methods OpenMew Method Lookin Biacore Methods Name Type hodified EE Affinity in solution kMelhod Builder 3 28 20H8 Calibration Free Concentration Melhod Builder 3 28 20H8 GST Kinetics Melhod Builder 3728 20H8 Inlect and recayer Melhod Builder 3 28 2008 Kinetics heterogeneous analute Melhod Builder 3 28 20H8 L1 liposome capture Methad Builder 3 28 2008 LMWy kinelics Method Builder 3 28 20H8 LM screen Melhad Builder 3 28 20HB NTA kinelics Method Builder 3 28 42008 Sinale cucle kinetics kethod Builder 3 28 2008
73. Control Sqmple R1A1 E060327 Thermo Rack Positions Raagent Rack 2 9 2 gh Deen Well Microplate 5 OOOO i 1 J J 060101010 01 e e OOOOOO 0 1 148 Negative control 148 Negative control 148 Negative control 148 Negative control 148 Negative control 148 Negative control 148 Positive control 148 Positive Cnntrnl 148 Positive control 148 Positive control 148 Positive control 148 Positive control 14 A amp nalyte amp 148 A amp nalyte amp 148 nalyte amp 148 nalyte amp 148 A amp nalyte amp 148 A amp nalyte amp 14 A amp nalyte amp 148 Analyte amp 148 A amp nalyte amp 148 A amp nalyte amp 148 A amp nalyte amp 148 A amp nalyte amp 148 nalyte amp 148 A amp nalyte amp Menu Automatic Positioning hutomatic Positioning Change the order in which the samples are positioned by ordering the regions The first region in the list is positioned first TyDe Control sample Control sample Control sample Control sample Control sample Control sample Control sample Control sample Control sample Control sample Control sample Contr
74. Kp 1710 10 5 PBS HEPES HEPES PBS DMSO DMSO 5 DMSO DMSO 10 0 05 Surfactant P20 3 1 DMSO DMSO 0 22 um
75. 060327 Thermo Rack Positions Reagent Rack 2 Type Sample 1 3 E 14 Negative control Control sample 4 i 148 Negative control Control sarnple i 148 Negative control Control sample ww 2 E 148 Negative control Control sample i 148 Negative control Control sample E 148 Negative control Control sample s 22 148 Positive control Control sample F i 148 Positive control Control sample 96 Deep Well Microplate 1 148 Pasitive control Control sarnnle 148 Positive control Control sample 148 Positive control Control sample 1 148 Positive control Control sample 11 i 148 Analyte A Sarnnle i OOOCOOO 148 Analyte A Sample i 14 A amp nalyte amp Sample 0 148 nalyte amp Sample 2 148 Analyte amp Sample 148 Analyte A Samnle E 148 A amp nalyte A Sample i 4 148 Analyte amp Sample 0 i 148 Analyte A Sample 2 148 Analyte amp Sample 148 A amp nalyte amp Sample 148 A amp nalyte amp Sample 148 A amp nalyte amp Sample 148 A amp nalyte amp Sample t hn CT lt Back Next gt Close Menu Automatic Positioning hutomatic Positioning Change the order in which the samp
76. Finish Result Plot Plot Result Plot Result Plot Edit File Save qs File Export Result To Excel Copy Graph Paint WordPad File Save Evaluation Method As Biacore T200 26 1 File gt Open File Apply Evaluation Method
77. 5 ul min 100 ul min CFCA IgG CFCA IgG IgM CFCA CFCA 4 11 molmzs Xx km Y D2 xf km 0 98 Xx 0 3 x hzxwxl D
78. 5 10 Thumbnails Quqlity Contro Thumbnails 4 Rejected Cleared Flagged Flag Accepted E Biacore T200 2 Kinetics Affinity 41 Thumbnails Status Tools gt Hide Rejected Reject
79. nsluded blanks 1 Calihbratinn free concentration Included blanks Include Cycle Flow ul min Temp C Zoom lock 1 hh ki i J 1 Nr 9 Im kk a wl yi W WM fh I N ll Include Include OK 4 18 CFCA CFCA Initialrate RU s 0 2 15 RU s lt 0 2 RU s gt 15RU s
80. Toolbar Ghat Har hart Bar ha 34B78 1113 2 pHHH2338 39B9BH H CwElE Humber Sample i WarFarine i ample _Warfarine canditign_baselne 8 _5ample 8 3 Warfarine 8 1 525 Cnrditinn lewl Sample Warfarine Sample Wafane 25 Sample WarFaringa Cnrditinn_lewgl g condition lewel 3 solwent 1 Sampls WarFarine colvent 2 Sample Wiarfarine solwent 3 Sample SS Warfarine E71 Controlsample 0 iti Curve Tools Tnnls mF FOU Ew Fk Color Ew k 1 hnwi Cunlumr Labes Biacore 1200 2 Kinetics Affinity 31 2 Kinetics Affinity Kinetics Affinity scree Kinetics Affinity
81. Browse Open es Binding Analysis Injection Sequence Detecton Flow path wv Chip type Capture SAMPLE 1 S ample Enhancemenl Regeneration REGENERATION 1 1 Biacore T200 2 1 Detectlon Flow path 2 1 4 3 Chip Chip type Capture 2 4 Sample
82. Tool bor MMg Create Affinity Screen Name Affinity Screen Curve Tuhe Corected Temperature Samples Include Sample i Curve Fc 4 3 corr Fc 2 1 corr Fc 4 3 corr Fc 2 1 corr Include Fc 4 3 corr Fc 2 1 carr Exclude All Fc 4 3 corr Fc 2 1 corr Fc 4 3 corr Fc 2 1 corr Fc 4 3 corr Fc 2 1 corr Fc 4 3 corr Fc 2 1 corr Fc 4 3 corr Fc 2 1 corr Fc 4 3 corr Name Curve Type Temperature Table Include Biacore T200 2 Kinetics Affinity 43 Affinity Screen Fit Settings gt Taols Clear Flags Sample D 1 Liqand parp15 Status IE Cleared gt Tools r Thumbnalls Hesults Summa Response Concentration Plot View vy Arrange By rv Zoom lock 1 parp14 Display Blanks Display Blank Subtracted
83. 5 SE standard error 20 SE 20 0 5 50 nM 4 21 Evaluation Explorer I DU Backspace Einetics Affinity Biacore T200 5 121 5 Biqcore H AG AH AS
84. Ranking Mode Ranking Result Plnt Curve Name Fc 2 1 corr Assay Step Purpose lt Dwerlawy lt ucle lt Hyerlau gt rj Tools v Result Plot Yalue Y Yalue Rankinq Rankinq yalue Sample Zoom Lock 5 3 06 Lowl 1 negative 5 3 H69 Law 1 negative 7 93 2 Low 1 STOSD2Z 8 D 406 Low 1 T_5 9 7 i 1 Tb 12 6 14 low 1 A_2 13 2 15 Low 1 A 3 14 ssd Law 1 A 4 Control sample 16 DOD 0169 Low 1 A 5 Sample 17 2 ol 1 Aa 7 18 3 52d Lowest Points DD negative 19 3 528 Lowest Points nenatiwe ZI 97 7 Medium 2 SsTOSDZ 21 2 30 Low 1 A 8 22 24 4 Low 1 A 9 23 111 edium I 2 1 24 1 697 Low 1 B_1 20 0 20 25 need low 1 B_2 Cycle 26 Lawi 1 B_3 Rieport Paint amp djustment Fanking Cutoff urye fitting 27 0 471 nl 1 B 4 28 533 Law 1 B_5 29 10 55 Low 1 B_6 Nane 0 Name re 30 1 724 Low 1 B 7 Ranking Edit i 31 6 108 Lowest Points negative Cut off Delele LOWesL Points 32 6 Lawiest Points 0 negative 33 Low 1 STOSDZ 34 16 Z2 Low 1 B_8 35 0 617 Lowl 1 B 9 36 3 Low 1 BE_10 a
85. Pooling Auto Poolingo_ Yes OK Automatic Positioning Biacore 1200 150 6 Eject Rack Rack tray port OK Eject Rack Tray Rqck Positions Next es Method Builder Prepare Run Protocol Tahorma i 1 Prepare Run Protocol akga sure the correct sensor chip 1s docked aka surm all sarmples headents are loaded in the rack and microplate according to the Rack Positions setup wials should ha sealed with rubber caps and microplate with adhgsiwg fn Flace the buffgrfs1 on the left hand tray and insert the correct tubing s see below ntal Standhy after run wl nsa buffer nA aka sure there is fresh w
86. OpenfNew Wizard Template Surface Preparation lookin a M thods And Templat Immabilization pH Scnuting SE Immobilization Name Type 2 0221 Kinetics Affinity Methods and Templates Open Browse Open Tes Kinetics hffinity Injection equence Cetectior Chip Flow path 4 3 Chip tupe Capture Regeneration Earr Ower Le ses 1 Biacore 1200 3 57 Detectlon Flow path 2 1 4 3 Chip Chip type Capture
87. Copy Graph Paint Word Pad Export Curves Finish Screening 2 Anyscreen Edit Menubar File gt Save qs Toble Export Thumbnails Export All Graphs And Table Thumbnails png Table txt Export Biacore 1200 52 2 Kinetics Affinity M
88. Browse Open LE Immunogenicity Screening Run ype Tn detect antibodies hnumd to u in the sample samples man he acidified to release the rhndies ard Iheh henutralized imrnediately before measuremert Ho do nu marnt to perform the measurements Direct analysis detects only free antibodies cidification and neutralization detects antibodies in the presence of excess drug OAcidification and neutralization Next Biacore T200 7 159 Immunogenicity Screening Injectinn Sequence Detection Chip Flow path Chip type SE wv Ligand capture ATPLE Sample EINHANCEITEINT Enhancemenlt REGENERATIUN 1 Hegeneraion 2 REGENERATION 2 1 Enhancement Regeneration Next lmmunogenicity screening SET Corditionng Hun conditioning cycle Lnrtact img ma ls Mumhgr of inections Startup Fun startup cucles Solutior tartuplpos control Mumher of cycles 3
89. uffer settings Runnina buffer SelpHun fter run 5 Specify analysis temperalure after rur Data Collection rate 1Hz Detection Multi 1 2 3 4 Sarmple compdrtment temperdture 4 45 C 25 C Concentration unit Buffer settings After run Assay Steps Biacore T200 6 143 Method Builder Main yeryiew Surface conditioninq eneral Settings Canditioning Canditionina 2 times as entered Wariable Settings Inject and Recoyer sample Inject and Recower 1
90. A RU vs C SO 100 1S0 2 250 Time A A Slope A Slope vs C 6 A A B A ecun esseseS9e Calibration Curve WIEHLIENIIII RU EBn E30 En 57n 540 510 amp 480
91. Enter value lt Convert diffusion caefficient from temperature T to 20 C Termperature T 3 D attEImheraturE Hg 1 1 rs CONVERT Diffusion coefficient qt 20 C 3 4de 11 Mn 20C 20 Biacore T200 96 4 4 2 1 Toolbar Run Method Menu bar Run gt Method 0penMew Method Ii Melhods nd Templates Modiied Biacore Melhods Biacore Methods 0pen 0pen New Method Lookin Biacore Methods Name Type Madified Affiniy In solution Melhad Builder 3 28 2008 Calibralion Free Concentration Melhod Builder 3 28 20HB ST Kinetics Melhad Builder 3 28 2008 Inject and recnyer Mel
92. 3 100 RU 10 000 RU DMSO 3000 RU DMSO Biacore T200 3 55 3 2 Biacore T200 Evaluation Software DMSO DMSO 1 x y
93. Empty min capacity 79ul Control sample Mix SCSSSOO 238 Individ 1 Sample 1 1S 238 Individ 10 Sample 4 O OOO 238 Individ 13 Sample 238 Individ 18 Sample 238 Individ 19 Sample 238 Individ 2 Sample 238 Individ 22 Sample 238 Individ 3 Sample 238 Individ 7 Sample wv ul Next 7 6 Menu Export Positions Menu Saqmple Position Import Biacore 1200 7
94. 2 2 Tools Remove Ranges Biacore T200 2 Kinetics Affinity 45 OK Remove Ranges Zoom lock owe data from injection start HH to injection start D 0 s and from injection end 1 2 s toinection end D 0 s The selected ranges will be rermowed frorm all curyes in all curye sets ill be cleared for accepted curwe sets pala eowed w_ J ea Thumbnails Tools gt Select Datq Remove Selection OK Affinity
95. 5 4 Run Stop Run Biacore Tzhh This will stop the run stop Run Runistopped Fimishimn Current cncle please wall bort cucle hn tlT Bre _ Ctrl Breakl _ Biacore T200 Evaluation Softwqre Biacore 1200 5 131 5 2 Evaluatton Biacore T2nn Evaluation Software Thermndynamics_antibody ys antigen blr 3 Curve Name Fc 2 1 me Assay Step Purpose lt Dwverlay gt Cycle lt Hyerlaw gt Sensararam dAll sensararams RU Sensorgram z Plot nnn nnm Lock a Baseline 5ample gi Binding level gi Binding
96. Finish Evaluation Explorer Sensorgram 1 nd ll sensnrgrais Flot a Easeline Sample Ontrols stahility 7 Oncentration nalyss li La Cnncehtratinn ha Biacore T200 4 7 RL Biotin 1Bnn 14nn 1gII Exxrlude Curwe THOU Exclude Cwcle 5hnwu 5Ensnrnram gn Lahels EH Scale Copy Goraph Export FS a Gridlines 2 nn LE YYYWYWYW WW z 2 A ED Hi ToD 12D Exclude Cycle Calibration Table REsHUTI E RU 138H
97. 110 4 Eject Rack Rack tray port OkK Eject Rack Tray Rack Positions Next Method Builder Prepare Run Protocol Prepare Run Protocol ake sure the correct sensor chip IS docked Make sure all samples amp readents are loaded in the rack and microplate according to the Rack Positions setup yials should be sealed with rubber caps and microplate with adhesive foil Place the butferts on the left hand tray and insert the correct tubing s see below intel Standhy after run will use buffer A Make sure there is fresh water in the water bottle nn the right hand tray If necessary empty the waste hnttle befanre start of the run Estimated run time Th 14 min excluding conditional statements temperature chanqes and standby flow Estimated buffer consumptior J Buffer a 1 lg 100 ml J 3 plus 65 ml day for Start
98. Toolbar Evaluation Explorer ororam jpiot Baseline AbsResp Binding level Baseline RelResp Binding stability Baseline RelResp Binding to reference Binding level Work qrea Evaluation Explorer Biacore 1200 7 169 Toolbar mmunogenicty Screening Screening Regioni settings Settings Curwve Fc 3 Hepart Point enhance_leve wv Riesponse Tupe Helalye Hespanse Heainns amp Baundaries Heaian Name Number of Regions Boundarw 35 gw Ce Settings Curve Fc 3 Report Point 2
99. Adjustment For Controls Use aqdjustment for controls 4djustment fnr Enntrnls Flnt dmitian Hghnrt point bindinn Response tupe Hglalwe Response arable Cucle stment settings se adjustrment for controls coom Lnck Positiwe control STOSD2 Menaliwe corntrol hedgatiwe Fitting unchinn Linear Pnlnmnmial 1nn Rua Before adjustment Fc 2 1 corr After adjustment Fc 2 1 corr ha mm E Eg 3 Positive control Negative contro None Set zero level 5 Fitting function Fitting Biacore T200 20 1 Linear Y qX b q b Polynomial Y qX lt bX c q b lt c
100. 4 Fitting Y 100 Y 0 OK Result Plnt Curve Name Fc 2 1 corr Assay Step Purpose lt Dwerlawy rj lt ucle lt Hyerlau gt rj Taols v Result Plot X Yalue Y Yalue Sample Zoom Lock 5 0 negative 5 0 negative 7 db 29 STOSDZ 8 2 562 T_5 9 2 846 T 6 12 9 239 A 2 13 5 229 A 3 14 8 638 A 4 Control sample 16 3 3Z6 6 SDM 17 3 292 A 7 18 DD negative 19 0 negative 2 101 3 STOSDZ 21 1 221 A 8 ZZ 2 9 23 114 8 A 10 24 5 241 BE_1 25 3 73 B_2 26 7 889 B_3 Fieport Point Adiustment ankina Eutaff Curve fitting 27 4 673 B 44 28 5 49 BE_5 Blank Sublraction ed head 29 15 63 B 6 Subtraction Tupe Nearest Blank 30 3 357 B_y Malecular weight dustment 31 n ti Molecular weight amp diustment negatve ER RTO djiustment For Controls ss omi Positive Control name STH8U2 33 100 7 STO802 Adiustment Far Controls Y Negative Control name negative 34 22 31 B 8 Fitting Funchion Linear
101. RNN NN EE se me ee ss me se ss ss EE Biacore T200 200 7 7 14 Menubqr LEdi Curve Name Fc 1 44 Assay Step Purpose lt Dwerlay gt Cycle lt Dverlay gt ensorgram JA sensorgrams RU Sensorgram a Plot 75nnn oom Lac ga Baseline Sample nr ga Binding stability H gi Controls enhancement 7oooo Se nso rg ram win d OW gi Controls stability ga Enhancement Peport Point Table BSnnn Hepart Point Tahle Evaluation Explorer og Cortrol Samhle amp Te 55000 Samhle Work area Startup Sn nn 1000 1200 1400 1500 Menubar Toolbar Evaluation Explorer ororam jpiot Baseline AbsResp Binding level Baseline RelResp Binding s
102. Settings Report Point Settings Report Pnint ettings Calculate rshnrse at position seBconds before inection stop mh Idnw sECnTHs Aapply To Selected Llgared Flaaed chinn will nt apply In accepted or rejected seres 4 Apply To OK Biacore 1200 46 2 Kinetics Affinity Settings Fit Settings Fit Settinps nd 5hleadp atg Hmtn plp To 5electg Cleared Flagged clio ll not apply to accepted or rejected seres Model 2 3 Parameters Fitting Apply To Selected Thumbnails Cleared Cleared Flagged
103. lt Lurwe Hame Fc g lt ssan Step Furhnsg HHywarlaus i lt Luclg xsHwerlar Znnm Lock Buffer BurFer hurFer negatiwe hnedativwe STOSDS TS T_5 Tr 1 Control sarnple A 5 5TIRIE Startuh Tt TiB atiwg EE 5 TiE tiwE 3 5TOBHg 7 9 5 31 8 1 5 BE_1 8 1 B 2 3 B 3 5 1 B 5 BE 5 1 5 B 5 5 5E Br Cwws Name Fr2 1 lt Assay Step Purpase lt Dvea Assoy Step Cycle lt Dvellyy lt Walue WWalule Walug VWalue
104. 5 Ok Y Median filtering Flot dhniinn Report point hindinn Hgshnnse tnne Relatie Respohnse hd edian Filtering settings se median filtering zm Lack ROSE T amls Tatal points 128 bowe rangs lnperlmt 1H In range Ps Lnwuer lirnit 5 IS Sethngs Eielow range 2 do width 1 Before filtering After filtering Lm Lm Lm lm Lm ml ka 1 Y Upperlimit Y 0 1 Lower line OK Biacore 1200 1 23 Tools Yalue Y Yalue Sample 5 3 069 negati
105. 1 4 1 Merged Injection 1 2 Analysis temperature Sample compartment temperature 10C Double Mix Angqlysis temperature 25 C_s Sample compartment temperature 10C 10C guanidine HCI Biacore T200 156 7 5 Biacore T200 Merged Injection 0 12 M HCI 0 1 Surfactant P20 1 3 M Tris HCI 0 05 Surfactant P20 pH 8 5 Tris HCI
106. 2 4 Sample Regeneration 1 or2 Carry Over Next gt Ts Kinetics Affinity Setup Lnndilinnim Hun conditiorning cwcla Contact hmg sl Mumher of inections 5tartun Huh startup cucles Solutior buffel Number of cucles Slent correction Huh solwernt correction Humber of inections 8 Fepeat after sample cucles Conditioning Solution contqct time s Number of injections otartup Solution Biacore T200 58 3 Number of cycles 3 Solvent correction EO RO TT I Number of injections
107. 5 8 mm2 ng Time Wd bsFiesp a LR lnne RglRsp Easeline 4 2 Il OD HI Yes baseline BBNN 5 11739 494 DD8 2 ad gg5n 5 dO 23803 19927 8B4 Biacore T200 7 153 7 Anti drug Antibodies ADAs Biacore T200 ADAs Drug ADAs Merged Inject Double Mix
108. Evaluatton YBiacoreT200 Eyaluation Software Concentration nalysis blr Sensararam Kineti ay ae ao Evaluation Explorer 1 x Remave Edit Tools v Sensorgram nd All sensorarams RU Plot 44000 Znam Lock a Baseline 5ample gi Binding stability gi Controls stability Hepart Point Table mm Heport Point Table 43000 43500 42500 42000 Calibratinn 41500 Cortrol Saimhle Saimhle 41000 Startup 40500 40000 39500 39000 4 5 Keyword table Tools gt Keyword Table Edit Chip Information 2 ww wv ww NF gt NN JN _N iii i i Biacore 1200 4 83 4 6 Menubar Too ba USB sensorgrams ER as urve Name Fc
109. Save qs Methods and Templqates Bia Users Don t Save Save Results From Run hs Savein E23 T100manual ku Hecent Dacumenls Desktop Mu Documenls 9 Mu Computer w My Network Biacore 1200 Immobilizatian of PrteinA blr manual blr pHscauking blr reqeneration check blr reneneratinn check_wizard hlr surFace parformance_wizard blr File name CFCA WW Cancel Save as type Result file Pl Save in File name Save T Hiacorg T2O00CGonirol sofhware Gh Example t 1 FR Et em Rn Took He EE er i cyck Fwes mrisnro am Fc L Lcck am SS hl Ediri RE 1 risal brding hiring Sieart R11 iocieion Fey Trempeahe 25 1 CC r Sm Irn Slat RA lr Flies EE TI F Bselre 1d Emmons in cele 3 Yah sislTg lah Fi ratisl EinHT sa lepPurpse gn Erdimn DydeType ample Sammlm_1_Blark ni a SanwMs_
110. Clear Flags Flag 2 2 Results Summary On Off Rate Map Table Kinetic Screen Fit Settings Taols Clear Flags Sample 2994 Ligand ref oxid Ritux Status Acceptecdl Tools v FPP Thumbnails Fiesults Summary Sensorgram Table Columns Zoom lack On OFF Rate Map a 5 gt 1 7T 71 gt BI GU 1000 1200 1400 1E600 Display Blanks Display Blank Subtracted Samples Included Curwes Flow Contact Time Diss Time ul min s s 180 0 300 0 1e 3 In n1 Off rate kd log 5 07e 04 and Parameters 1 37e 05 54e 77e Quality Control Report Hesiduals Parameters 5 GBG 9 94e 04 Kinetic constants are within instrument specifications 5 73e 04 1 91e n5 5 1 Kinetic constants appear to be uniquely determined 1 ls No sigrificant buk contibutions RN found 2 38e 05 a significant bulk conthulians HI faun 1 14e 056 es 3
111. 4 Sarmple Regeneration 1 or 2 Carry Over Next Thermodynamics Setup Landitionina Hun condilionina cycle Solutior Contact time ls Number of iniections Startup Hun startup cycles Solutior buffer Number of cucles 3 Snlyent crrectinn Hun salyent carrection Number of injections Repeat after sample cycles Temperatures Analysis temperatures Sample compartment temperature Temperature a a Conditioning Solution contact time s Biqcore T200 Number of injections Startup Solution Number of cycles Solvent correction 5 125 3
112. 69 3 11 DMSO DMSO DMSO Solvent correction Extrapolate olvent torrection Exrtrannlatinn Range E wtrapolation Fiange fw amis TODD RL 10 OK RU 10 zoom loc 3H 2HHH 15HH 1000 5nn n SH 1000 1500 2000 25nn Response Ref RU Biqcore T200 1 Kinetics Affinity
113. Report Point binding Assqy Step Sample RelResp RHJ 2 1 10 Tools gt Custom Report Points Add Fhdd Custom Report Point Heport point ld stability2 Window 5 sg Position the report point 2n seconds after sample 1 injiection stop Calculate response relative to report point Cycles Apply Ta Selected Assay step purpose Appl To Selected Cycles ld Cycles OK Gustom Report Points Custom Hennrt Foints hisay FED DUFrpOsE stabilitws gH seconds after stop oF alihratinn Injgrtinn 5amhls 1 Sample Iinjgrtinn Control sample Biacore T200 30 1 1 11
114. Biacore T200 Evaluation Softwqre Biacore 1200 7 165 7 1 1 Immunogenicity Screening Toolbar Run Method Menu bar Run gt Method Te 0pen7New Methnd Laok jn Methods And Templates Name Biacore Methods Show importable wizard templates 0penMNew Method Lookin 100922 Name Tupe kodified Immunaaenicity Screening creening 12242010 Show importable wizard templates Open Method Builder Moin Overview Biacore T200 Biacore 1200 166 7
115. ul 7 12 Menu Export Positions Menu Saqmple Position Import Biacore 1200 198 7 Immunogenicity Screening Prepare Run Prntncnl Tahoma 10 BH U Prepare Ruh Frotocol ak sure the correct sensor chip 1s docked akg sure sarmples eadehts are loaded in the rack and microplate according to the Rack Positinons setup ials should be sealed with rubber caps and microplate with adhgsiwg foil Place the butferls on the left hand tray and insert the correct thi
116. Save in File name Save Standby flow Biqcore T200 Evaluation Softwqre Biacore T200 4 4 Run gt Stop Run Biacore Tz A This will stop the run Stop Run Runistopped Fimishimn cirrent cncle please mal bort cucle hn LlT Bre _ Ctrl Breakl _ Biacore T200 Evaluation Softwqre Biacore 1200 82 4 4 1 2
117. R1A1 R1A12 12 060327 Thermo Rack Positions Reagent Rack 2 Type Sample 1 3 E 14 Negative control Control sample 4 i 148 Negative control Control sarnple i 148 Negative control Control sample ww 2 E 148 Negative control Control sample i 148 Negative control Control sample E 148 Negative control Control sample s 22 148 Positive control Control sample F i 148 Positive control Control sample 96 Deep Well Microplate 1 148 Pasitive control Control sarnnle 148 Positive control Control sample 148 Positive control Control sample 1 148 Positive control Control sample 11 i 148 Analyte A Sarnnle i OOOCOOO 148 Analyte A Sample i 14 A amp nalyte amp Sample 0 148 nalyte amp Sample 2 148 Analyte amp Sample 148 Analyte A Samnle E 148 A amp nalyte A Sample i 4 148 Analyte amp Sample 0 i 148 Analyte A Sample 2 148 Analyte amp Sample 148 A amp nalyte amp Sample 148 A amp nalyte amp Sample 148 A amp nalyte a
118. 4 17 Menubar Toolbar Evaluation Explorer Work area Menubar Toolbar Evaluation Explorer ooram Po Baseline Work area Biacore T200 Binding level Binding stability Binding to reference Assay Step Purpose lt Dverlayy Cycle lt Dverlay gt Sensorgram window AbsResp Baseline RelResp Bqseline RelResp Binding level Evaluation Explorer nnrertratinn analysis Toolbar 4 115 CaliBratian reg sa Calibration free concentration elect Samnles Create Expand all cucles hnwu original sehnsorgrams se reference subtracted data Fr 4 3 1 Hu iml Protein A HH5 mnusg IgG 1 HI Fc 4 3 Fr 4 3 Touse ld x1i6400 Fc 4 3 Dilution Flow Initial rate OC ratio TEnnp fartnr tu
119. Biacore 1200 7 171 Biacore T200 Evaluation Software Ex1 Screening merged blr Screening Fle View Evaluation Tools Window Help sensoraram Plot BarChart A Kinetics AFfinity gt Concentration Analysis lt Thermodynamics Immunogenicity gt Evaluation Explorer 1 xiRemovye Edi pg Report Paint enhance_lewel Baundaries not bound lt 35 RU bound gt 35 RU Adiustments for controls No adjustments Sensorgram JJ Allsensorgrams Plot a Baseline Sample ga Binding stability RU a Controls enhancement 120 Neg control 0 ngiml 4 not bound a Controls stability Pos control 1 naiml 8 bound ee i Eo Serum 3 8 nat boun eb ot Table 100 Sample 0 5 uaiml 9 bound a3 Screering Sample 0 5 x100 drug 1 bound Serum 3 9 not bound 80 Sample 0 5 Haiml 7 bound Neg control uiml 65 nat bound Pos control 1 ajml 5 bound Serum 3 5 nat bound En Sample 0 5 Haiml 2 bound 1 Serum 1 5 nat bound Serum 1 9 not hanund Serum 1 2 not hound 40 Serum 2 3 not bound Neg cantral uiml 7 not haund Pos control 1 jml 1 bound Serum 2 4 not haund 20 Sample 0 5 Haml 3 bound Serum 2 8 nat bound Serum 2 8 not bound Sample 0 5 haiml D bound Sample 0 5 ug ml 1 bound Neg control 0 uiml 1 not bound Pos control 1 ajml 4 bound 20 Serum 3 9 nat bound 0 10 20 30 40 5n B0 70 80 an 100 Sample 0 5 x
120. guanidine HCI 10C Cycle Run List Next Biacore T200 162 7 Immunngenicity Screening Cycle run list 5ample 1 Solution Startup Immunogenicity Screening Rack Positions Fieagent Fiack 2 8 es db ell Microplate OO I I I I 0 OOS OO I I I 1 1 OOS ee OOS ee OI ML LL Startup pos control Startun nos controly Startun nos controly Neg control 0 ml Pas control 1 ajml Sample 0 5 ug ml Serum 3 Sample 0 5 ug ml Sample 0 5 x100 drug Serum 3 Sample 0 5 pg ml Serum 3 Sample 0 5 uiml Serum 1 Exactly 50 Ne control 0 pgyrml Exactly 50 Neg control aiml Exactly 5H Ne control 0 u jml Exactly 50 Neg control 0 ugjml Exactly 50 Ne control 0 uajml Exactly 50 Ne control 0 uajml i Exactly 50 Me control 0 ugjml Exactly 50 Pos control 1 uajml Exactly 5H Pos control 1 ugjml Exactly 50 Pos control 1 ug ml Exactly 50 Pas control 1 ugjml Exactly 50 Pos control 1 ug ml Exactly 50 Pos control 1 uajml Exactly 50 Pas control 1 ugjml Exactly 5H Pos control 1 uajml Exactly 50 Startuptpos cnntral Exactly 50 Startun nos co
121. 113 4 2 2 Evaluation BiacoreT200 Eyaluatinn Software C Documentsand Settings DbiacoreMWesktonMBcT100y2MCFCA_ProA lgG hme we Sensorqram _ A Kineti ini 2 i i Evaluation Explorer 1 x Remove Edit ei Sensorgram All sensorgrams Sensorgram Plot gi Baseline Sample Binding lewel gi Binding stability gi Binding to reference Hepart Point Table mm Heport Point Table Concenlralion Analysis Frotein mause la Zoom Lack Blank Saimhle Start up Time 4 16 Keyword table Tools gt Keyword Table Edit Chip Information se Le ee 1 eG EE HEIEHEHEHEEEEEE 1 1 1 Biacore 1200 114 4
122. 177 Immunogenicity Confirmation Prepare Run Protocol Prepare Run Protocol Make sure the correct sensor chip Is docked ake sure all sarmples amp reagqents are loaded in the rack and microplate according to the Rack Positions setup yials should be sealed with rubber caps and microplate with adhesive foil Place the buffers on the left hand tray and insert the correct tubing s see below Notel Standby after run wl use buffer A Make sure there is fresh water in the water bottle on the right hand tray fnecessarY empty the waste bottle befnre start of the run Estimated run time 12h 3min excluding conditional statemenls temperalure changes and standby Flow Estimated buffer consumption A Riunning bulfer atleast 3UH ml plus 55 ml day Far standhy ater run Start Save qs Methods and Templqates Bia Users Don t Save Save in File name
123. 1 1 Enhancement 2 Regenerdtion 1 or 2 Next Tes Binding Analysis etup Lnhdiinhi Hun cnnditianinn cycle so IlhUhv WI Contact hrng sl Mumher of inections Gtartup Run starktup cucles Sun Mumher of cycles 3 Startup Solution Number of cycles 3 Biacore T200 1 3 Next 1 1 Sqample 4 ss Binding Analysis INjection SEequenese Faplure SAIPLE 1 Sample 3 PLE 2 Enhancem
124. Cortrol sample Samhle Salyert carrectinn Startup 1 6 Keyword table Tools gt Keyword Table Edit Chip Information LT 4 a 4 le a ee re a ee a sn a ee a le W ee ee ee ee ee W ee ej W ee EE me i se 9 ee NN NN a 1 Biacore 1200 1 11 1 7 Menubqr P i AM TOO ba RE sensorgrams Edit Rh urye Name Fc 2 r 4 Assay Step Purpose lt Hverlay gt Cycle lt Dverlay gt Sensorgram window Evaluation Explorer gt Work aread Menubar Toolbar Evaluation Explorer ororam
125. 40 s l min s 4 75 Cancentratinn Lnalysig 7a lihratinnm ry Galihratinn ur aalyte name Bepeat calihratin ewery 20 um 54V Run last alihratinn points Concehntration Calibration Curve Analyte name Run first Repeot calibration every 1 Run last Calibration points Concentration 6 2 Next Biacore T200 76 4 es Conc
126. Inject and recover Open Method Builder Main Overview BiqcoreT200 Biacore 1200 6 141 Method Builder Main 2 Assay steps Concentration writ na ml LO Data collection rate 1Hz a Sample compartment temperature 25 C Detection Multi Inject and Recover Cucle Types sample Inject and Recower 1 time as entered Settings for assay step Surface conditioning ee IT NN Wariable Settings EpandAl rd General settings Biacore T200 ethod Builder Kain Beneral Settings gt Data collection rate Detection Sample compartment termperature Assay Steps Hz Vary with analysis temperalure ucle Tupes rere Miscellaneous wm Concentratiorn unit
127. 1 Merged Inject Biacore T200 154 7 Adjusted response 8 8 rituximab ug ml rttuximab ug ml Ant ritirmah umlin serum Ant ritimah 0 25 uimli serurr Anti rituxirmab 0 ugimlin serum Anti rituximakb 0 5 ugimlin serum nt ritueirmah 0 5 ugirmlin serurm e Ant rituirmakh 1 uml in serum a Ahnti rituxirmabh 1 ugirmlin serum Anti rituxirmab 2 ugirmlin serum 2 ADAs 3 ADAs Double Mix Drug ADAs ADAs Double Mix
128. ADAs ADAs ADAs ADAs Merded Inject Drug ADAs ADAs 1 2 3 ADAs Merged Inject rm ra Merged inject a PH 2 3
129. F 20 0 2 40 50 go 100 1 140 Cyelc number be OK Biacore T200 48 2 Kinetics Affinity Rax Lineqnr Polynomial Fitting Y Control Rmax x Entered Rnax value x RmQx for series 1 Rmax for series 2 series 1 x Series 2 CyCle no Steady State Affinity Constant Rmax Multi Site 2 Fit Settings nd 5teadu 5late it Lnnstant Hmaw Ilulhi Site Control Hmar RUADD Da diust Rimas for cnntrnl 1s turnhed nl dust Fimas Fnr Corntrols ustrnerts ill pnlp to sgrlgs Ihat use Cortrol Rrmas apply To Selected
130. 1 1 Kinetics Mw knm Bivglent Analyte CFCA Christensen Anal Biochemistry 1997 249 p 153 Sigmundsson K et A Biochemistry 2002 41 p 8263 Biacore T200 92 4 CFCA 5 000Da Ke 107 gt Kg gt 5X104M 1s 1 150 kDu 5 000 RU CFCA 0 5 50 nM 1 ug ml 280 nm 10 4
131. Biacore 1200 7 199 7 4 2 lt Evaluottion Biacore T200 Eyaluatinn Software Userst17 00 verz_ demo datatimmunogenicitytEx6 Stability bme Plot Bar Chat A Kinetics Affinity a 5ensararam _ Concentration Analysis Thermodynamics Immunogenicity Evaluation Explorer ds All sensnrgrams 3 x Remave Edit Curve Name Fc 4 3 Assay Step Purpose lt Hyerlaw gt rj Cycle lt Dverlayy rj ensorgram All sensorgrams RU Sensorgram I Zoom Lock Plot 16nnn oom Lac gi Baseline 5ample gi Binding stability i Binding to reference Hepart Point Table mm Heport Point Table i Immunogenicity Stability BI Time TM 9 TT Keyword table Tools gt Keyword Table Edit Chip Information
132. Save Standby flow Biqcore T200 Evaluation Software Biacore 1200 178 7 7 2 2 Evaluatton Biacore T200 Evaluatinn Software C MBia UsersVT100 yer2_demo dataMimmunogenicity Ex 4 Confirmation bme A Kinetics AFfniy Cancentration Analysis Thermadynamics Immunanenicity Et All sensnrgrams x Remowe Edit 4 5 Curve Name Fc 3 rj Assay Step Purpose lt Hverlaw gt Ccle lt verla gt rj ensorgram Al sensorgrams RU Sensorgram Po 65nnn gi Baseline 5ample gi Binding stability i Controls stability Hepart Point Table mm Report Point Table a 9 Sensorgram _ Plot ul Bar Chart Zoom Lock Cortrol Sarmple Saimhle Startup 7 7
133. Run control samples Run control samplels every 10 20 Next Biacore 1200 7 175 Immunngenicity Confirmatinn Cycle Run List Assay step name Sample 1 Solution Sample 1 Mix solution Startup Startuptpos contral Buffer Startup pos controly Buffer Startuptpos contral Buffer Neg control Burfer Pas cnntral1 pgimly BuFfer Indiyid 3 Burfer Individ 3 drug Indiyid 3 Burfer Individ 3 drug Individ 13 Burfer Indiyid 13 drug Individ 13 Burfer Indiyid 13 drug Indiwid 1 Burfer Sample Indiyid 1 drug Control Sample Neg control Buffer Control Sample Pos cantralt1 pgmly Burfer Sample Indiyid 1 Burfer Sample Indiwid 1 drug Individ 10 Euffer Individ 10 drug Individ 10 Burfer Individ 10 drug lt Next immunogenicity Gonfirmation SystemPreparations Frime before run Momalze detector Temperature settings halusis temperature FUI Sample cnmhartrmEnt temperature FLI Sample compartment temperature 10C 10C
134. 134 5 Kinetics Affinity Select Data Create Curwes urye Fc 2 1 Ligand antibody 10ug ml pH5 Sample antigen Temperature ee tul min s s 6 Fc 2 1 3U 7 Fc 2 1 3 8 Fc 2 1 3 9 Fc 2 1 3 10 Fc 2 1 3D Blank Subtracted Sensorgrams Zoom lock 0 Kinetics OKinetics Affinity Fit Kinetics Create urye Fc 2 1 Ligand antibody 1Hun ml pH5 Sample antinen Temperature add Fi Current Fits Mode 1 1 Binding y No fits performed Description Parameters Nh sr edt aa he yah ee A a Ct TN re de de er Cancel Biacore 1200 5 135 Model 1 1 Binding Fit Kinetics 7 Affinity Fit Rinetics Ureate urwe Fc g Ligand arntibody 1Hu ymlnHH Sample amen Tempneralure dC H Fit Lurreht Fits ode 1 1Bindina 1 1 Bindin Wo oS antigen 8 5 nM Huality Lnntral Report Hesiduals Parameters Finish Evaluation Explorer
135. Enhancement 2 Riegeneration 2 2 Enhancement 2 Biacore T200 190 7 7 3 3 Evaluation 3 od EK 1 Eyaluation Explor 1 Curve Name Fc 2 1 rj Assay Step Purpose lt Hverlaw gt Cycle lt Hyerlaw gt Sensorgram JA sensorgrams RU Sensorgram Plot 2500 a Baseline Sample gi Binding stability i Binding to reference Hepart Point Table Hepart Point Table Zoom Lack 7 10 Keyword table Tools gt Keyword Table Edit Chip Information i iii
136. Extra wash ADAs 2 ADAs 2 human IgG lambda human IgG kappa Desorb 1 Desorb 7 157 and Sanitize Cut off SD x 1 645 95 Mire Sluis A R Et q 2004 J Immunol Methods 289 1 16 wizard Immunogenicity Screening Immunogenicity lsotyping Merged Inj
137. Table Table Include Cycle s Plot QC Plot Exclude Result Plot Plot Plot QC Plot Biacore 1200 1 15 Result Plot QC Plot Plot QC Plot nn Tools Windnm Help orrection ng Sensorgr rd Sensorgre fae Bar Chart Ft Finetics Finity Concentration Analysis_ CE ame Fc 2 1 oc Pt 4 Assay Step Purpose lt erlans 4 Cyele we a ssult Pot Binding level TO Plotname Y Axis X Axis Flot Settings Plot name HC Plot Plot tupe Report Point ws Wariable Report Foint ws Report Foint
138. Eject Rack Truy Rack Positions Next es Concentration Analysis Prepare Run Protocol Prepare Run Protocoal lake surg the correct sghSnf EhI 1s dnckgd Make sure a samples fBadgnts are Inadgdin the rack and microplate according tn the Rack Positions Satuh yials shnuld be sealed with ruhhgr cahs and microplate with adhasiwg fall Flacg the buffgrfs1 nn the left hand tray and insert the correct tubels ntal Standhy after run wl nsa butfer a akg sure there is fresh water in tha water bottle on the right hand traw If necessary Empty the waste hnttle before start of the run Estimated run time dh 54 min encluding cnnditinnal statements temperature charnges an stahndby Flow Estimated butfer consumptior Running buffer 1 A At least 300 ml 3 1 plus 65 ml day for standhy after run Start Sqve as Methods and Templqates Bia Users Don t Save
139. Flagged 2 3 Affinity 3 Steady State Affinity 1 1 Binding Affinity Steady State Affinity Constant Rmax 1 1 Binding Rir Biacore T200 2 Kinetics Affinity 47 Fit Settings 5tmadu State hnitr Lnnstant Hmas Cantral imar RUZ10nDal diust Hmas For cnntrnl is urned nf diust Hmaw Fnr Controls HHustments wl annln to serlms Ihat use Corntrol Hmax Phlp To Selected Cleared Flagged ctior will nt ly to accepted or rejected series Rr Rr Rmgx Control Rmax RU 100 Da 30 RU 300 Da
140. Number of replicates Times Cycle Types Biacore T200 6 145 Inject and Recover TE Method Builder Kain Dwerview re Description of selected cycle type Inject and Recnyer The Inect and Hecnwer cpcle tupe renulates sample inlectinn washing of the Heneral Settings Candihanrr swstem and recowery of he material that is captured on the sensor surface withirn one cycle the sample Injection and recowvery sequence Ca be Assay Steps repeated up to 14 times The total sample wolume maximum roughly 30 EE micraliter will be equally divided over the number of repetitions Note that salts and detergents present in recovery deposition wash solutions and running buffer can reduce sensitivity In mass spectrometric MS analyses on the recovered sample ucle Tupes Variahle Settings Verification Commands Report Points ettings for Inieck amp ndHiecnyer 1 Sample solution Sample Method Wariables
141. oo 0 0 0 0 GO i Cs Lee ws ul 4 13 Menu Export Positions Menu Sample Position Import Biacore 1200 4 109 4 14 Control Sqmple
142. Cycle Types mM Methnd Builder Main _ oswss Beneral Setings Condeonng es sns Locsues vaioe emo Verification Comards Report Points 6 PP Melhod Variables Evalualion Variables Get property as yariahle SetpRun Sample 1 Sample solution el Contact time FD Contact time f s Acid solution wait after acidification s Neuralizalion salution Dissaciation time s fter acidificalia 0 ks Flow rate pl min inn saln 1 3M TRIS 05 5 mw hzs4 Saqmple Type Double mix Acid solution Neutralization soIn Double mix Biacore T200 7 167 7 1 3 lt Evqluottion 5 Curve Name Fc 1 Assay Step Purpose Hyerlau gt Cycle lt Dverlayy rj ensorgram Nd All sensorgrams RU Sensorgram Pl rs000 gi Baseline Sample i Binding stability i Controls enhancement i Controls stability Enhancement Hepart Paint Table mm Rep
143. mi 2 Hl 4 Kp kg k Kp ka k Biacore T200 122 5 vant Hoff Kp5 AG RTInKo AG AH TAS InKko AH RT AS R AS R intercept on Y axis a AG kJ mol z In Kp R T K _AH R slope Kp M A AH kJ moD AS UU K mol 1 T RTInKn AHT TAST ACp T To TACslnl AC k
144. 2 Biacore T200 70 3 i Evaluation Explorer Plot Baseline Sqmple boseline 42342 42332 RU 5 Cycle number 10 42338 Carry Over co_binding co_boseline Startup
145. Ranking low i Fle View Evaluation 5 a Sensorgram Plot miBarChart 9 Kinetics AfFinity 3 Concentration Analysis gt Thermodynamics 3 Immunogenicity gt Evaluation Explorer 1 x Remoye Edit Sensorgram JA sensararams Plot ga Baseline Sample Binding stability ga Controls stability Hepart Point Table mM Feport Point Table Immunogenicity 3 Confirmation Confirmation 2 ww Biacnre T200 Evaluation Software C MBia Users T100 yer2_ demo dataMlmmunngenicityEx 4 Confirmation bme Gonfirmation Tools Window Help Settings Curve Fc 3 Report Point stability Sample cycles Mised with no drug Boundaries low lt 150 RU high gt 150 HLU diustments for controls No adjustments RU Sample 5 1 P Ranking 400 a Individ 13 no drug 151 1 i 31 Individ 18 no drug 228 5 Individ 19 no drug 227 6 350 Individ 22 no drug 391 0 Individ 14 no drug 226 2 Individ 21 no drug 389 3 Individ 17 no drug 226 8 300 Individ 16 no drug 226 4 Individ 20 no drug 389 1 Individ
146. cs 165 2 167 7 2 ADAs 172 7 i 172 Biacore T200 CN fd 178 7 3 ADAs LL 182 7 3 1 Merged Inject 182 7 3 2 Double Mix NWCYyYOO 188 FE MM i 190 7 4 ADAs LLL 193 kOe A eA 193 7 4 2 L 199 Biacore T200 1 1 1 1 1 Toolbar Run Wizard Menu bar Run gt Wizard ssay Finetcs a trnty EiElimEEmEIUEE Lnncenlralinn Analysis Thermndunamics Assay Binding Analysis New Methods and Templates Open
147. 11 Biacore T200 7 191 7 11 Menubar Toolbar en Curve Name Fc 1 44 Assay Step Purpose lt Dwerlayy Cycle lt Hyerlaw gt ensorgram JA sensorgrams RU Sensorgram a Plot 75nnn oom Lac ga Baseline Sample nr ga Binding stability H gi Controls enhancement 7oooo Se nso rg ram win d OW gi Controls stability ga Enhancement Peport Point Table BSnnn Hepart Point Tahle MMM MI Evaluation Explorer sem 55nnn Work area Cortrol Samhle 1 Saimple amp Startup Sn nn 1000 1200 1400 1500 Menubar Toolbar Evaluation Explorer ororam jpiot Baseline AbsResp Binding level Baseline RelResp Binding stability Baseline RelResp Binding to reference Binding level Work q
148. Control Rmax X_100 30 RU X 100 300 Da 30 RU 100 Do Rr M Ws nlytg aayte ContralRrn nx x To Adjust Rmax For Controls Ru Control Rmax RF ES RU 100 Do RU 1UU PO 6 mm Adjust Rrmax for controls lc Flot dghrion Report poirt bnding Hesponse type FAIU Venable Cycle Adustment setthrgs Use Au Rimax for cortrole Adustmerite WI apph to al sees th Lse Cortrol Rmax Fosfvc cartml B2u kontol 3231M 7 Fitting function Lineer Poknomia RU Control responses Fc 2 1 TT 35 Te CS 5
149. jpiot AbsResp Baseline RelResp Bqseline RelResp Binding level Evaluation Explorer Baqseline Bindind level Bindind stability Bindind to reference Work area Biacore 1200 12 1 5000 1 data File gt Append Result File Solvent Correction Quality Control QC Result Plot Plot QC Plot
150. s sELI ns Hieport Foint arlahlg Hesponse Tupe Fielatiwe Riesponse MS Finish Plot QC Plot Tnnls nlnr By Table Culumn lgrular Weight dijnstment Binding level resyj Molecular Weight Adjustment Keyword Table Y 100 X RU Da Biacore 1200 16 1 Tees Capture Adjustment Result Plot Plot Result Plot Sensotgram i Plot lt il Bar Chart a kinetics AFFinity Lurwe ame Fc 2 1 co RU 1 100
151. 6 co 35 5 725 B_9 kMiedian Filtering 35 G 2 B_1n FR Median Filtering Y X Y non binder potential binder X Y Y Biacore 1200 1 21 RL Result Plot 2 15 E10 8 median EE 5 mm LTTH LM 5 1n 15 190 195 2 2 5 31 215 22 H 225 23 wrlE X Y 0
152. BH 7 U Prepare Run Protocol akga sure the correct sensor chip 1s docked aka surm all sarmples headents are loaded in the rack and microplate according to the Rack Positions setup wials should ha sealed with rubber caps and microplate with adhgsiwg fn Flace the buffgrfs1 on the left hand tray and insert the correct tubing s see below ntal Standhy after run wl nsa butfer nA aka sure there is fresh water in the water bottle nn the right hand tray If necessary Bmpty the waste bottle before start of the run Estimated ru mg 1 h mm encluding conditional statermernts temperature changes ad standby flo Estimated buffer consumptior 1 Running buffer 3 3 lgast HH ml plus 5 mday tor standh after Fun start Sqve qs Methods and Templqates Bia Users Don t Save Save Results From Run hs Savein Ti00manual 92 mmobilzation of antibody blr Immobilization of Prteina blr hy Hecent manual hlr Documents
153. Biacore T200 H9 3 NN OU 6 DR 9 F OD HH
154. Check that sensorgrams haye sufficient curvature Pw HI EE 1 1 1 1 On Off rate Map Map Copy Graph Paint Word Pad Export Curves Finish Screening Kinetic Scteen Edit Menubar File gt Save qs Biacore T200 42 2 Kinetics Affinity Toble Export Thumbnails Export All Graphs And Table Thumbnails png Table txt Export Affinity 2 2 2 2 Affinity
155. Cleared Flagged to wll nt apply to accepted or rejected series Steady State Affinity Constant Rmax Control Rmax Rmax Rmaxz Adjust Rmax For Controls Biacore 1200 2 Kinetics Affinity 49 Fitting Fit Plp To Selected Llaargd Flaqged cio ll nt hl to accepted or reelected series Kinetics 7 Affinity Calculating Results Samples Fitting sample b of 21 Lurye Ligand Fitting OK Sample Chiz RUz Fc 2 1 corr Fc 4 3 corr Fc 2 1 corr Fc 4 3 corr Fc 2 1 corr Fc 4 3 corr RR Fc 2 1 corr Fc 4 3 corr Fc 2 1 corr Fc 4 3 corr Fc 2 1 corr Fc 2 1 corr Fc 4 3 corr Fc 2 1 corr Fc 4 3 corr Fc 2 1 corr Fc 4 3 corr Fc 2 1 corr Fc 4 3 corr Fc 2 1 corr Fc 4 3 corr 0 584 0 371 2 52 139 D0632 0 0475 FI ry y Fitting Toble
156. High Rmax Biacore 1200 2 Kinetics Affinity 37 Tools Remove Ranges Remove Ranges nnm Inck Hgmnwg Hala Irnm IlEclinn start HH sl In necton start fs ahnd From inectiorn end HU to InIEBchinr end H H sl The selected ranges will he rermnwed frorr a tatys will be cleared for accepted ciee sets Existing fits will be rermowed ok Thumbnails Tools gt Select Datq Sample 3117 Ligand ref oxid Rx Status C Clear Time Displsy Blanks Display Blank Sublacled Samrles Biacore 1200 38 2 Kinetics Affinity Blank Subtracted Sensorgrams Remove
157. Include Cycle Biacore T200 88 4 4 8 RL Biotin 1Bnn 14nn 1gII Exxrlude Curwe 1UH Exclude cycle 5hnwu Sensorgram Fn Lm Lm Rative Re Lahies BI Scale Copy Goraph 4nn Export Curwes Gridlines nn Lengrid I 2 DI FH sl 1 1 Contentration inl Exclude Curve Calibration Table Lurye lt Hyerlaw gt RU Biotin 1Bnn 14nn 1200 1000 GU Relative Response BU UI Parameters 2nn lt Hverla gt 20 n 2n 40 ED 8n 1nn 120 Concentration ncitnl Include Curve Biacore T200 4 9
158. Ri Rz k k tu ti 50 ti1 2 In2 kg Biacore T200 A 1 Al 8090P 00692003CPOR4 DFN 153 154 156 172 180 181 182 193 AQUSUMeSDE TGC OS ee i 19 169 170 ANdVSIS tSMOeFU NE eS 5 60 77 125 147 155 161 186 196 B BI Ve i bi SS BING TGRE EE NC ii 70 Buhlek se s CC 97 142 C SUDOUONRCUNVS SN 95 84 85 COV Nh i he iN hi 57 65 70 124 CC i 91 92 93 116 120 CONC eMAUOMLANOVN SS GG GAN 72 84 86 91 CO SA 57 124 143 146 CO ES i 4 59 60 76 89 161 174 196 CUSIOMIReDORP OL RC Ni 29 89 UO NG i GG 23 25 157 160 192 D DoCoMo A i i 97 142 Deebos tS SO UO i i 145 DO 0 fi 153 154 155 156 157 165 166 188 189 E ERCIUOE CUE a i ei 88 EC CUE Ci 14 87 CUOGDO NS 67 ERO WS Oe eC A 58 125 UD OO CC ihc A 69 H HOVESOUUON Gk 4 74 126 MU ILS 15 158 165 172 182 188 193 NCIS UU Ni i 88 CO CS i i eS Si 14 87 RC UDO ON CREE CERO ct CEE EN 145 Biacore T200 K Ri Gi Go i i 91 M Mared eC 153 154 155 156 157 158 182 184 193 MUU i i i cc 74 R ROORIG Se 23 24 171 181 RecCOVeN SO ic 145 S IO
159. SD Slope RUAs LRSD FelHesp FRU Baselne Sensorgram late lt J All sensorgrams binding 02437 Plot binding HIB2UE ga Baseline Sample binding 0 1026 ga Binding leyel binding D 04047 D 04043 D 0974 U UB585 1545 U IH818 1 1 HI25E2 123 U IUE455 1641 UI5382 0 1879 D 03872 0 1998 OD OG7E 0 2045 1028 1785 UEB55 0 1799 03735 0 1777 D D3E08 0 1537 D D303 0 1702 D DOSE81 1548 0315 0 278 1533 1333 D 02218 135y 1423 18Hy D DOG287 1B55 U 14E2 0 1918 D 01823 0 1905 D D1102 0 1539 D 01717 1B8E In1EE1 1488 U 3215 0 1749 D D02334 0 2192 D 1079 0 1858 0 009351 binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding binding a Bindina stability a Binding to reference i Controls binding Controls stability eport Point Table mM Hepart Point Table Solvent Correction 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 Biacore T200 1 29 Fc
160. sOOOOOOOOOOOO P sOO OOOOOOOOQOOO gt 66 9 9 0009000900eeee OOOOO OOOCOOOOCOOO Reqion Color Drientation Anchor Rack ial 5i i First Sort By Control sample Cyan i Column Bottom leFt Sample Smal Ascending EE CarkElue i Column Bottom left Sample Small Ascending Crimsan EColumn Battam left Sample Smal Ascending Oelow i Column Bottom left Reaqent r Large Ascending Salyent correction buffer a Elue EColumn Battom lat Reagent Small Ascending gt Pooling Auto Pooling ves OK Automatic Positioning Biacore 1200 5 129 Eject Rack Rack tray port
161. Binding Analysis Control Samples Control sample definition Hun control samples Repeat control samplels every Control samples Control sample id 1 Cnntral 2 Repeot Control Samplels every OK Next 1 2 Excel Excel Excel txt Kinetics ffinity System Prenaratinns Frime before run Mnrmalze detector Temperature settings halusis emperature LC Sample compartment temperature LC Prime Normalize Anqlysis temperature 25C Sample compartment temperature 25C
162. Contact time Contact time s OM 5 Dissociation time s Flow rate plermin Flow rate ls yariahle Piedp NO Mi sw Cycle types currently in Method Sample Commands Sample1 Settings for Sqmple1 Type High performance contqct time s 36 Dissociation time s 5 Flow rate ls variable Setup Run Variables Flow path Both Commands Regeneration1 Regeneration solution contact time s Flow rate ulMmin Flow path Both Variable Settings Biacore 1200 4 103 Method Builder Main Define yariahle handlinn for each amp ssay Step General Settings sample Define all yalues at run time Assay Steps Define all walues in method Deline some values in method
163. Regeneration Next InmmmumngEnirity Screening Set Lnriditinnin Hun cnndilinninn cuclg Lnrtacl mg sl Mumher of inections itartup Fun startup cycles souion Humber of cucles 3 Next Biacore T200 7 195 ImmmumngEnicity Scereening Injertinnn Parammeters Sample Contact time ls lawurate lmm Dissociation hmg s Hgneneralinn Siolutior 100 mM NanH High wiscosity solution Contact Im FIanwrate mml Stabilizatinn perind In Eshra wiash after inection wt 5 mk aH 120 Next Immmmumngenicity Screening Samnls Sample table Sample id Hu nrdgr s enlergd Handam Run order Next Biacore 1200 196 7 Immunngeni
164. Tools wv 5 D 5 In 7 15 33 8 0 4239 9 4531 12 1 472 13 833 14 1 376 16 In 53 1 17 0 5247 18 In 19 In 2D 16 15 21 0 1948 22 4 417 23 18 3 24 0 8359 25 0 5949 26 1 259 27 0 7455 28 gyG1 29 2 494 3 In 5358 31 In 32 0 33 15 08 34 3 552 35 1 074 36 1 41 negative negative STOos02 T_5 T_6 2 A 3 4 A 6 A 7 negative negative 5TOSI2 A 8 A 9 aA_10 B_1 negative negative STOS02 B_8 B_9 B_1n wv 1 19 Capture Adjustment RU RU boseline Cqpture_baseline Adjustment For Controls
165. f h w Biacore T200 4 12 CFCA 20C 324 3x10 1 fxmreixMu173 m2 s f T rel 20C Mw Do Biaqcore Biqcore T200 Biacore Biacore T200
166. pHscouing blr regeneration check blr reqerneratinn check_wizard hlr surFace parFormance_wizard hlr Mu Documents 39 My Computer File name Thermodynamics_antibody ws antigen S kp Network Save as type Fesult file blr Biacore T200 64 3 Saqve in File name Save Standby flow Biacore T200 Evaluation Softwqre 3 6 Run Stop Run Hiacore Tzoh A This will stop the run Stop Run Runistopped Fimishimn current cncle please mal bort cucle hn LlT Bre Ctrl Breakl
167. 1 Merged Inject Toolbar Run Wizard Menu bar Run Wizard LE WTndikihn Pl FHIllir 5urfaca Preparation Immahllzalinn pH 5cnuhin Immahilizahinn EE Assar Development eathods nd Templates Nam Buffer Scouting H723 5uifacg Peformance 317 a Lnntral Experiments 8 gt Kinehics Linked Haarhinns 4 2 Regerneration Seouting n9n521 Fihetics Mss Transter SS Kmehics Fi Bimdirmn halpslg Lnrcenlrahinn nalsls Thermndumamlcs uhnnehlcllu Immunnnerlcltu CrEETIh Immunanenlcitr Lnnhirmahinn Immumanehnlcltu lsntuhin mmunogenicity lsotyping RunType Tn detect antibodies hnumd to drug In the sample samples mau be acidihed to release he rhndies and thern neutralized immediately before measuremernt Hnwu do nu arnt to perform the measurermernts Direct analysis detects orly free antibodies CO cidification and neutralization detects antibodies in the presence of encess drug Merged Inject Acidification and neutralization Direct analysis Next Direct qnalysis
168. 1 3 M 4 M NaoOH pH 8 5 Surfactant P20 1 Double Mix 0 12 M HCI 0 1 Surfactant P20 1 0 M Tris HCI 0 05 Surfactant P20 pH 8 0 Tris HCI 1 0 M 4 M NaoOH pH 8 0 Surfactant P20 1 10 mM Gly HCI pH1 5 2 5 30 20 isopropanol 40mM NaoOH
169. 15 no drug 226 2 250 Individ 3 no drug 564 9 Individ 1 no drug 61 7 1 OE Individ 10 no drug 148 8 200 Control Sample Individ 7 no drug 148 5 Mix solution no drug Individ 2 no drug 58 6 Individ 8 no drug 146 8 8 150 Indiyid 9 no drug 146 5 Individ 6 no drug 57 5 Individ 4 no drug 55 1 Individ 11 no drug 145 0 100 Individ 12 no drug 144 1 Individ 5 no drug 57 2 bk Neg cantral no drug 68 2 9 5n Pas contral 1 pgiml no drug 234 7 Neg control no drug 56 7 Pos contral 1 ajml no drug 227 1 0 Me contral no drug 5n n 0 10 20 30 40 50 ED 7O Pos controlt1 uaiml no drug 223 0 Cycle Humber Neg control 45 9 ow Immuno conF 15 bmpn Paint high high ADAs Biacore 1200 182 7 7 3 ADAs 7 3
170. 2 4 Assay Step Purpose lt Dverlay gt Cycle lt Hwerla y Sensorgram window e mm Report Point Table Evaluation Explorer Work area Menubar Toolbar Evaluation Explorer ooram piot AbsResp Baseline RelResp Bqseline RelResp Binding level Evaluation Explorer Baqseline Bindind level Bindind stability Bindind to reference Work area Biacore 1200 84 4 Toolbar Concentration analysis La Lsinn calibration Using calibration IGoncentration Analysis Create Calibration Control Samples Samples Calibration urye Settings Flow Cell Fe 1 Report Paint stability Response Tpe Fielative Hespanse Use precedina calibralion curve Use ayerae calibration curye Use calibralion trends Calibration uryes Calibration Trend
171. 21 Above Cut off D_1 133 2 Abawe Cu of D_2 SEE Show Sensorgramts 0 3634 Below Cut ofF D_3 Report Point Adiustment Ranking Cut off Curve fitting 13 04 Above Cut off D4 see 19 62 have Cut off D_5 Exclude Cycle s Blank Subtraction Malscu 46 89 Above Cut off Copy Adjustn 0 8927 Below Cut off opy Table Malecular weight amp diustment Positive Neo Le gt Capture Adiustment Fitting F 2 787 Below Cut g 99 22 Above Cut F No saturation klediar adiustment For Controls 4 Upper Lowier i Median Filterina Windo lt gt High Rmax 3 16 Above Cut off Remoye annotation Biacore 1200 28 1 1 9 Curve Fittino Result Plot Curve fitting Linear 4 pqrameter Fitting At Smo Pupoeew Smwe Cc vets Result Plot 1 38DSA DAmno BS 4 enz60e9sifoname3e Dansyiamne SupremeGe 3 7 Bensenetulfoasmide Bensenetulfoesmde 3 Bensenetulfoesmde 2 Benzsenetulfoesmde 6 Bensenetulfoasmde 1 5 Bensenetulfosnsmde 0 Bensenetulfoesmde 3 Bensenetulfonsmide 0 Benzenetulfoesmde Tools 2 Benseneculloe smde Bensenetulfoesmde Benzenetulfonsmde Sulpride Bensenetulf onsmde Damino SA Dam
172. 3 Temperature Temperature ci Biacore T200 138 5 Next Thermodynamics Results Create Lalculaled Thermandunamic Faraprete want Hnff Fithinn Funchinrn yant HanTf Fittinn unchinn IE min g 5 dB d doB d d dB 3 3 5B d dB dB d 5B d 5GB 3 BB 1T 1 Paramelers Chis dL Ts Ewring dissociation 1 2 135 3 5B 3 5B 5B 5B 55B 3 3 3 3 3B 5B 5B 3 5E 3 55E 3 BB 3 IT 1 IT 1 PFarameters Farameters lopes 18Hr Ads Intercept 14 92 lds FF db lope BrBE ls Intercept 1 7 A FE HHHr lt Back Linear Non linear Fitinn Fctior lyant Hnl Fitinn Funchinn IE Inn Linear Farameter Name Farameter walue Emol a HAE ol Ta El
173. 3 times as entered 1 2 3 1 2 3 O Drder 1 1 22 3 3 OO Random Number of replicates times Biacore T200 3 4 99 Ts Method Builder Main J startup startup 3 times as entered te ns J sample sample sample 1 time as entered HHS sample 1 time as entered Before every 10 cycles Sample Number of replicates times Biacore T200 100 4 5 Method Builder Main _owveu en Start up General Settings Delele Startup Startun 3 times as entered Copy Assay Steps Sample Cycle Tupes Sample Sample 1 time as entered Move Up Wariable Settings Move Down Cycle Fiun List Assay step properties Base settings Name lank Purpase Sample Bmeto lsmps cycle type Assay step preparations Recurence BA meeasssspe ange Eye cycle Distribute 1 occurrences evenly Hun assay step once first Run assay step once last Number of replicates Temper
174. Biacore T200 7 183 Immunogenicity lsntyping Injectinn Sequence Detection Chip Flow path vy Chip type CM5 yl Ligand capture SAMIPLE Sample REAGENT 1 lsotyping reagents lse 2 vy reagentls per cycle REAGENT 2 REGENERATION 1 Regeneration 1 v 1 Use reagent s per cycle Regeneration Next immunogenicity lsntyping Setup Lnrndiinin Hun conditioning cycle Contact hmg Mumher of imgchnns 5tarluh Fun startup cnclgs nlulinn sErum 5Bred Mumher of cycles 3 wv 3 Next Biacore T200 184 7 Emmunogenicity lsntyning Injection Parameters Sample Lnnlacl fime ls Flow rate lmim lsnluhiri reagent Corntact fime ls Elow rate 5 lmiml Hgnernerahinn nluHinh mM NaLH High iscosity snluHinn Corntact time 3 ls Flawuralg lmm Stabilization perind np Etra wiash after inection wi 5 mM NaH
175. Capture wv Settings for Sample 1 Type LU Method Variables Ewaluation Wariables Insert Remowve Sample solution s wariable property as wariable Sample 1 Sample solution scphn Regeneration 1 Contact time Contact time s Regeneration 2 Acid solution Dissociation time 0 ls wait after acidification s i N Neutralization solution cid solution 120 mM HCL 0 1 P20 a Dissociation time s wait after acidificatior 0 Flaw rate min Neulralization saln 1 3M TRIS 0 05 P20 Flaw rale 5 min Flow path 1234 MM Extra wash after iniection with Stabilization period lp s sample Type Double mix Acid solution Neutralization soIn Double mix Method Builder Main Cucle thes Description of selected cucle type Screening General Settings Conditioning Assay Steps Cycle Types Wariable Settings Enhancement v Settings for Enhancement 2 Enhancement solutior antilgG Melhod Variables Set property as variable ime ED sehn un EE IO Enhancement solution FIOW Ial 10 min Contact time s hi Flaw rate plemin Flow path wv Fredip Extra wash after iniection with Stabilization period p
176. Kinelics Mass Transfer Assay Kinetics Affinity Bindinn amp nalysis Concentration amp nalysis Thermodynamics Immunnnenicity Immunaqenicity Screening Immunogenicity Isotyping Laok ir Methods and Templates Name Tupe L Immunogenicity Immunogenicity Confirmation New Immunngenicity Confirmatinn Injectinn Sequence Detection Chip Flow path 3 Chip type LEMS Liqand capture SAMIPLE Sample Enhancement REGENERATION 1 Begeneration 2 ww REGENERATION 2 1 Enhancement Regeneration Next Biacore 1200 7 173 mmunogenicity Ennfirmmatinn Setup Lnrdiinnimn Hun conditiorning cwcla Corntact ime ls Mumher of inections 5tartuh Huh startuh cucles Golutior Startuplpos cantrol umhgr nf cucles 3 3 Next Emmunagenicity Gonfirmation InjectionPara
177. Selection OK Settings Fit Settings Fit Settings 1 Binding J pply To Selected Cleared Flagged ti ill nt nl tn accepted or relected series Model Parameters Fitting Apply To Selected Thumbnails Cleared Cleared Flagged Flagged Biqcore T200 2 Kinetics Affinity 39 Fitting Fit Plp To Selected Llaargd Flagged cio ll nt apply to accepted or rejected series Fitting OK IKinetics 7 Affinity Calculating Results Samples Fitting sample 1 of 15 Chiz RUz lteralion 2 ChF 0 952 RUE ax r
178. Sinale sample series mouse IgG x1 5400 Fc 43 3 oe tu NN 40 SD ime S 0se Remove Selection Undo Biacore T200 Calibration free concentration Evaluatinn Result Create Dilution Meas Lonc M Furve factor Calc Conc Oc M 119 Temp Mw D ratio C Da m lt s 4 434E 10 1 714E 09 100ug ml Protein amp pH5 0 mouse IgG x1 6400 64nn 1 nnuqjml Pratein amp pH5 0 mouse IqG x1 1600 16nn Fc 4 3 Fc 4 3 mouse lgG x171600 Processing series 2 of 4 lteratior 48 Ma relative change 1254 kd 0 002900 Calibration free concentration Evaluation Result Create Expand cycles Fr 4 3 1HHuajml Prntein a H5 mnuse InG 1 hi hn 4 434E 10 Fr 4 3 1HHunjml Protein amp H5 H mouse InG x1J1EHni 15Ihn 1 71E TE mouse Il x1400 Fc 4 3 2 838E 06 2 742E 06 37 25 425 25 150000 150000 5 82Z6E 11 5 826E 11 Zoom lock 8 Flowe SUlmin Flow 10 Hlmin Lalr Lnnr th1 3BE E HL Temp D ratin C mirsh ey 2 15DODD 5 E
179. and others at un time Cycle Tupes Variable Settings alues for these variables will be defined at run time Command Command Variable t Flow rate ulimin E Fw D 2n C SeupRun Diution Enter walues for yariahISS defined in the method These yalues will be used for all cycles in the assay step Assay Steps Sample Define some values in Method and others at run time 5 Setup Run Variables Sample solution Flow rate ul min 5 l min 100 ul min 2 MW Do D 20 20 C Dilution Blank No n Biacore T200 104 4 ms Methnd Builder Main gt Assay steps Define wariable handling for each amp ssay Step General Setting Define all yalues at run time i Assay Steps N Define all wal
180. point enhance_leyel Riesponse type Fielative Hespanse Wariable Cycle Number diustment settings Use adjustment for controls Positive control Pos control 1 na ml Negative control Neg control 0 pom Ba Fitting function Linear Polynomial Before adjustment Fc 3 After adjustment Fc 3 h ho a mm Lm co mj Relativue Response enhance_level Adjusted U 40 EO gS0 40 ED 8n Cycle number Cycle number Cg Cea Adjustment settings Use adjustment for controls Positive control Negative control Fitting function Linear Polynomial Y 100 Y 0 OK
181. time as entered Cycle Tupes Cycle Hun List Assay step properties Base settings Riecurrence Name Surface conditioning Repeat assay step withir Conditioning Every 1 lt cucle Connect to Conditioning Distribute 1 lt occurrences evenly cycle type Run assay step once first Run assay step once last Assay step preparations Temperature las Bulfer As entered 1 2 3 1 2 3 Drder 01 1 2 2 3 3 Random Number of replicates Times 3 Injection and Recover Biacore T200 144 6 Method Builder Main Surface conditioninq Canditianina Conditioning 3 times as entered Yariahle Settings Inject and Recoyer sample Inject and Recower 20 times as entered SelupHun Cycle Hun List Assay step properties Base settings Hiecurrence Name J Repeat assay step wihir Purpose Sample Every 1 cycle 3 Connect to Inject and Hecnver 7 Distribute 1 lt nccurrences evenly cycle type Hun assay step once first Run assay step once last Assay step preparations Temperature Bulfer As entered 1 2 3 1 2 3 Order 1 1 2 2 3 3 Random
182. 100 drug 1 bound Cycle Humber Serum 3 5 not bound Rqanking Biacore 1200 172 7 7 2 ADAs ADAs ADAs 7 2 1 Toolbar Run Wizard Menu bar Run gt Wizard 0pen New Wizard Template Surface Freparation Immobilizalian nH Scouting Immobilization NE Assay Dewvelopment Regeneration Scouting 390521 Bulfer Scouting 030723 Surface Parformance 0d17 Contral Esperimens 31119 Kinelics Linked Heachians
183. 1_h E11 rmlial Eindmg Sample_1_lcww_rate 1 Erdimn Bammle 1 Lioard ama_1_Lmrari CE ammgls_1_Lirsr 1 ccc ammle 1 hu 1EETrn Gam 1_ Samph E53 TO P Fa ru rh pa a rh TI Temperathe 5 oC ile compart mernt bemhsraturs Ont ZS SC emt 2s CC Runnin TiHhy remaring me 4 ms Biacore T200 Evaluation Software Biacore T200 112 4 4 15 Run gt Stop Run Biacore Tz This will stop the run Stop Run Run Stnhpe Finishin Current Cucle please wa bort cucle hn tlT Bre _ Ctrl Breakl _ Biacore T200 Evaluation Softwqre Biacore 1200 4
184. 200 100 D Biacore T200 32 2 Kinetics Affinity 2 1 Keyword table Tools gt Keyword Table Create Kinetic Screen Name Kinetic Screen 2 Curve Tuype Helference5ubtracted ww i Temperature Samples Include Sample Ligand oxid Ritux reF nxid Ritux oxid Ritux reF oxid Ritux Include oxid Ritux ref oxid Ritux oxid Ritux ref oxid Ritux oxid Ritux ref oxid Ritux oxid Ritux ref oxid Ritux oxid Ritux ref oxid Ritux oxid Ritux TeF nxid Ritux Name Curve Type ReferenceSubtraction Temperature Table Include Biacore T200 2 Kinetics Affinity 33 Kinetic Screen Fit Settings gt T
185. 3 51 2436 1 113 5h1 8 1 113 5n5 HHH1 HZ 113 1 HE5 1 Empty iMix min capacity 114Ll Empty iMix min capacity 114Ll Empty iix min Capacity 114Ll Empty Mix min capacity 114Ll Empty iix min Capacity 11 Empty Mix min capacity 114Ll Empty ix min Capacity 114Ll Empty Mix min capacity 114Ll 113 51 2435 1 113 551 2435 1 113 SO5 DOD1 02 113 DI HE5 1 Empty Fix min Capacity 114Ll Empty Mix min capacity 114Ll Empty ix min capacity 114Ll Empty Mix min Capacity 114Ll Sample Sample Sample Sample alibratinn mix alibratinn Pi alibratinn Mix alibratinn ix alibratinn Mix alibratinn Mix alibratinn Mix alibratinn ix Sample Sample Sample Sample alibratinn Mix alibratinn Mix alibratinn Mix alibratinn ix wu sarnnle 1 Lnnr ngn 1 1 Empty Pix min canacity 114pl Canntrnl samnle Mix Emptyw iiz min capacity 114pl Cnntranl sample Mix Empty Pix min canacity 114pl Control samnls Mix Emptw Miz min Capacity 114pl Cnntrnl sarnnle Mix 113 5I1 3 1 113 5H1 9 1 113 5I5 InH1 3 113 PET 01 Sample Sample Sample Sample wf ul
186. 48 Negative control Control sample i 148 Negative control Control sample E 148 Negative control Control sample s 22 148 Positive control Control sample F i 148 Positive control Control sample 96 Deep Well Microplate 1 148 Pasitive control Control sarnnle 148 Positive control Control sample 148 Positive control Control sample 1 148 Positive control Control sample 11 i 148 Analyte A Sarnnle i OOOCOOO 148 Analyte A Sample 148 Analyte amp Sample 0 148 nalyte amp Sample 2 148 Analyte amp Sample 148 Analyte A Samnle E 148 A amp nalyte A Sample i 4 148 Analyte amp Sample 0 i 148 Analyte A Sample 2 148 Analyte amp Sample 148 A amp nalyte amp Sample 148 A amp nalyte amp Sample 148 A amp nalyte amp Sample 148 A amp nalyte amp Sample t hn CT lt Back Next gt Close Menu Automatic Positioning hutomatic Positioning Change the order in which the samples are positioned by ordering the regions The first region in the list is positioned first Region Color Urientation Anchor Rack Yial Si i First Sort By Control sample cx an i Column Bottom leFt Sample Smal Ascending Sample EE CarkElue i Co
187. 8 Positive control Control sample 11 i 148 Analyte A Sarnnle i OOOCOOO 148 Analyte A Sample i 14 A amp nalyte amp Sample 0 148 nalyte amp Sample 2 148 Analyte amp Sample 148 Analyte A Samnle E 148 A amp nalyte A Sample i 4 148 Analyte amp Sample 0 i 148 Analyte A Sample 2 148 Analyte amp Sample 148 A amp nalyte amp Sample 148 A amp nalyte amp Sample 148 A amp nalyte amp Sample 148 A amp nalyte amp Sample t hn CT lt Back Next gt Close Menu Automatic Positioning hutomatic Positioning Change the order in which the samples are positioned by ordering the regions The first region in the list is positioned first Region Color Urientation Anchor Rack Yial Si i First Sort By Control sample cx an i Column Bottom leFt Sample Smal Ascending Sample EE CarkElue i Column Eottom left Sample Small Ascending Startup Crimson i Column Eottom left Sample Smal Ascending wash Oelow i Column Bottom left Reagent Large Ascending Salyent correction buffer a Elue i Column Bottom left Reagent Small Content Ascending gt
188. C MO 133 OMEC OR eC ON tO ih 58 66 68 69 125 T 00 077000002 72200000 P27 2022 0700077007027 2 07 007 202 203 U SV 86 9 61 99IHU9 NIMIUI AE 84 V VO i i i i i i 122 DR i 153 156 182 183 185 Nik i i i 139 144 145 146 152 PURE 91 92 3 RA NR cc A a 65 70 lt LD TM 30 koo Mii 169 180 PP 71 75 84 85 87 88 91 NKKRKKANN KK UI 91 153 RE i 4 5 12 19 20 24 50 60 70 76 89 155 157 160 161 169 174 180 196 0 156 Biacore T200 1 ocm0 2 91 0 A 1 18 28 31 42 46 53 158 172 KEK 193 RE ROSS RSS 122 138 7 74 153 172 BL 1 153 156 71 AA ii 121 122 54 55 202 203 GGG 122 138 5 70 154 155 161 5 5 7 14 15 18 Se 5 20 24 47 48 70 155 156 170 173 OO 93 94 Wc 91 92 NR SR 91 92 116 KNWWQW K 53 31 54 55 58 65 66 67 68 69 125 24 169 201 40 DCRR OM 29 89 Biacore T200
189. Da Sample 1 D 20 burfer burfer buffer Euffer Euffer mouse InG x1 6400 150000 5 09E 11 mouse IqG x1 6400 150000 5 09E 11 mouse InG x1J16 150D000 5 09E 11 mouse InG x1J16 150000 5 09E 11 Burfer Buffer mouse IaG x1 400 150000 5 n9E 11 mouse IaG x1 400 15nnnn 5 n9E 11 mouse IaG x1 100 15nnnn 5 n9E 11 mouse InG x1 100 150000 5 9E 1 1 gt Biacore 1200 108 4 Next Method Builder System Preparations Prime befare run Narmalize deteclar Prime Normalize Next Method Builder Rack Positinns Sample and Heaqent Hack 1 Type Sample1 Sample1D San Mw Da 20 C Sample Ss 118 Buffer 61 Buffer 118 Buffer 61 mause IqG x1 6400 Sample 150000 5 09E 11 118 mouse IqG x1 6400 5ampnle 150000 5 09E 11 61 mouse IgG x1 1600 5ample 150000 5 09E 11 i 118 mouse IqG x1 1600 Sample 150000 5 09E 11 61 mouse IgG x1 400 ample 150000 5 09E 11 1 Os CO 118 mouse IqG x1 400 ample 150000 5 09E 11 i 1 mouse IgG x1 100 ample 150000 5 09E 11 9 2 118 mouse IgG x1J100 Sample 15nnnn 5 09E 11 1 artu 208 buff O eo 725 ah pH1 5 Re 1 E01 OO OT J 6
190. EE 11 zh 15OODD ghE 1 1 anm lack Flow THmin Frted 12 FlInww Tin 13 Flnw 1 nrHlmin1 Frted 13 Flnwr 1HUUlimin CM Meas Conc Biacore T200 120 4 CaqlcConc M Meas Conc OC ratio QC ChiZ RU2 SE Megs Conc Meas Conc 4 20 Finish Evaluation Explorer Lnficertralin halusls libratinnF reelonc 4 20 CFCA Chiz
191. GE Heqlthcare Biacore Life Sciences BIQCOre T200 version 2 Instrument Handbook GE imagination at work 1 kk 1 Si hk 1 Ma 10 2 Kinetics Affinity 1 re 31 2sl Kineties fi 31 2 2 Affinity UL ee 42 3 ee 53 56 FF EM ai 66 a coc ci 71 4 1 71 1 NGAG Ga 72 2 AR SO 82 4 2 a 91 2 2 00 EE EK 96 ME A 113 5 kk 121 5 i 123 2 GG 131 6 ee 139 6 Sj SG 140 7 EK N OOOOOOOIOIOIOIOIIIIIEIEIEEI 153 158 7 1 1 Merged Inject NN Ea 158 7 1 2 Double Mix
192. InG anti_ IgG anti IqG anti_ IqG anti_ IgG anti_InG anti_ IqG anti IqG Biacore T200 186 7 Immmmunngenmicity lsntyhing system Prenaratinns Prme before run NHnrmalize detector Temperature seltings halisis emperature FI Sample compartment temperature E Bee lt Bsk New gt ges Analysis temperature Sample compartment temperature Analysis temperature Sample compartment temperature 10 10 a guanidine HCI 10C Cycle Run List Next Immunmogenicity lsotyping Rack Positinns E 73 AO Sample 4 amp Sample Sample Sample Sample Sample Sample Sample i Sample dB Wygll Microplate Sample Sample a ll 110060e ee OOOO Sample nO XX MN nt TJ eeliele So Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample lt Back New gt ces ul
193. J K mol To 25 C 8J Eyring Ka k k ksT exp AS R AH RT Ink T AS R AH RT inkB h k AS R In ka intercept on Y axis kg h AS OK moD R AH kJ mol KT T K AG kJ mol AH R slope 1 T Biacore T200 5 123 5 1 Toolbar Run Wizard Menu bar Run Wizard 1 OpenfNew Wizard Te mplate Surface Preparation nb Em Look ir Methads And Temnlates y Immobilizatin Assay Development Name Type Regeneratinn Scouting Thermadynamics Buffer Scouting Surface Performance Contral Experiments Kinetics Linked Reactions Kinetics Mass Transfer hssay Kinetics affinity Binding Analysis Cancentratian Analysis Immunngenicity Immur
194. Manual Edit Settings Result Plot Curve Name Fc 2 1 corr Assay Step Purpose lt Dwerlawy rj lt ucle lt Hyerlauy gt rj Taols v Result Plot Zoom Lack 6 3 69 Below Cut off negative 93 2 Above Cut aFf STOSDZ 8 18 3 52 Belaw MM negativwe 19 3 528 Below Cut off negative 20 97 hnve cuoff STO8 2 21 Eelow Cut off A 8 So Belawi Cut ofF T_5 9 0 223 Below T 6 12 51 Above Cut off A 2 13 2 16 Above Cuk off A 3 5 14 5 56 Above cutof A 4 Cartral sample 16 Eelow Cut off A 6 Sample 17 0 235 Below Cut off A 7 EE abowve A 9 22 24 1 23 1 Abowve Sa 1 24 1 697 Abowe Cut ofF B_1 25 0 186 Belawcutofl Baz 26 Abowe Cut off B_3 Hustment Hanking uoff Furwe fitting 27 0 4718 Below cwol B_4 hode 28 J Below Ct B 5 Nane Automatic Canal sample name negative ya 3 254 SD 1 583 w 1 ca Ranking Manual No of SD 3 31 6 108 Below Cut off negative Cut off Response 1 484 32 Below Cut aFf negative 33 1 ave ca STOSDZ 34 16 Z Above Cut ofF BE 8 35 0 6171 Below Cut off B 9 36 3 7B6 Abowe Cut ofF BE_10 S Table Cut off Cut off Below Cut off Cut off Above Cut off
195. Samples Included Curyes View Small Thumbnails Standard Thumbnails Extended Thumbnails Arrange By Affinity Screen Fit Settings gt Tools gt Clear Flags Sample B 1 Ligand parp15 Status E cleared gt Tools Thumbnails Riesults Summary Response Concentration Plot View Arrange By Zoom lock 4 Zoom lock Display Blanks Display Blank Subtracted Samples Included Curves Display Blanks Included Curves Biacore 1200 44 2 Kinetics Affinity Include
196. Set property as variable Contact time ls Flow rate Flow rate pl min Wash salulion Recovery salulian Wrash solution 5 Deposition solution Deposition solution yolume Inject amp ndHiecayer 1 Sample solulion RE 5 Contact time s Flow palhi Recovery solutior lIncubation time Deposition solutior SOmkd NHA4HCODS Deposition solution wolume ul Number of repetitions Sample solution contqct time 5 Flow rate umin Flow path 1 2 3 4 Wash solution Recovery solution 0 Incubation time Incubation time s Deposition solution
197. Sulfarilamide Sample 148 WWarFaring Sample 148 WarFarine Sample 148 WWarFaring Sample i 148 WarFarine Sample 95 Mgl icraplate 148 WWarFaring Sample 148 WarFarine Sample i 148 WWarfarine Sample 148 WarFarine Sample 148 WWarFarine Sample ip 1 148 WarFarine Sample 148 WWarfarine Sample 6 148 huFFer Startup 1 i 148 buffer Startup 1 1 1 1 1 3 145 buffer Startup i 541 ibufFer Reqenesration 393 5IOe DI5 Wash 1 1 1 1 1 OT 4151 50 DM5O Wash 4 1 1 1 1 Full i Solwent correctionl Soleent correction buffer A Full i Solwent cnrrertinn2 5nlwent correction buffer a Full i Saleent correction3 5nlyent correction buffer a i Full i Solwent correctiond Soleent correction buffer a 1 1 1 Full i 5nlyent corrections 5nlyent cnrrertinn buffer a H F Full i Solwent cnrrertinn5 Solvent correction buffer a gt u 3 4
198. alysis temneralure after rur Data Collection rate 10Hz Detection 2 DualMult DuQl 2 4 3 Multi 2 1 4 3 2 1 3 1 4 1 Sample compartment temperature 4 45 C 25 C Concentration unit Buffer settings After run Assay Steps Biacore 1200 Lcceseex SeupHun Assay ste properties Base settings Name startup startup startup 3 times as entered I sample sample sample 1 time as entered blank Sample sample 1 time as entered Eefore every 10 cycles Hecurrehce Repeat assay step wilhin Purpase Startup Ewery cycle Connect to cycle type Assay step preparations Temperature Bulfer Startup Distribute ncCurrehCBs evenly Hun assaw sten once first Hun assay sten nnce last Number of renlicates
199. amp 450 4on 390 3ED 330 300 270 10 20 3 40 SD BI Mil SD go 100 110 Concentration units TIl Biacore T200 72 4 4 1 1 Concentration Analysis Toolbar Run Wizard Menu bar Run gt Wizard ssay Finetics aFfinity Ea 1 3 Assay Concentration Analysis New Methods qnd Templotes Open
200. ater in the water bottle nn the right hand tray If necessary Bmpty the waste bottle before start of the run Estimated run tirme Sb mn encluding conditional statements temperature chahnges an standh flow Estimated buffer cornsumptior A Buffer a 3 least 1 ml hlus 65 ml day for standhy fter rum Start Save qs Methods and Templqates Bia Users Don t Save 2 ED Methods and Templates My Hecent Inakayama Documents ED Ti00 dema File T100manual ED Templates Desktop My Documents 9 My Computer File name KineticsAffinity 1 1 interaction bme SS Ky Network Save as type Biacare T100 Eyalualion Files bme Biacore 1200 6 151 Save in File name Save Standby
201. ature 1 gt times Bulfer As entered 1 2 3 1 2 3 Irder 1 1 2 2 3 31 Handam blank ODistribute occurrences evenly 1 Recurrence Number of replicates times Cycle Types Biacore T200 4 101 Ts Method Builder Main Dverview ETuh Descrintion of selected cucle type ee Fm Assay Steps cceTwpe Variable Settngs Yeriication Commands Report Points Caplure Settings for Sample 1 Tupe High performance Method Variables Evaluation Variables Sample solution buffer Sample solution Contact time 35 s Contact time fs Dissociation me s Dissociation time 5 5 Flow rate plemin Flow rale 3 MM min Flow palhi Eoth v Set property as variable Fraction lg gl of mis solution otabilization nerind after mis 0 Ezra wash after injeclion with Stabilization period lp Startup
202. city Screening Cnntrnl Samples Lnntral samrle delinlinh Hun cnnlral sampls sample cucles Corntrol samples Run control samples Run control sqmple s every Next Iimmnnngenicity Screening SystemPreparations Frim before run Mnrmalige detector Temperature setthings halsis emperature EC Sampls compartment temperature FLI Analysis temperature Sample compartment temperature Sample compartment temperature 10C 10C guanidine HCI 10C Cycle Run List Next gt Biacore 1200 7 197 Immunogenicity Screening Rack Positions Heanent Hack 2 73 D7 Sample B 73 D8 Sample 96 Well Microplate i 73 D9 Sample 199 serum Startup 946 100 mM NaOH Regeneration 803 50 mM NaOH wash Full 100 mM NaoH predip Predip
203. ded Curves Include Table Blanks from other sample series 3 Rejected Cleared Flagged Flog
204. der Detection Detection Flow path Flow path Next Biacore 1200 106 4 shethod BuildEr Variahles arahle yalues for nssay Step Sample EE mt 0 sampe kton Row rate Gwin ay 9 DO pton mnusa I G g1 4 15hHHH 5 nGE m 2 mause igG x1 6400 15HHHH 5 gE 11 3 mause igG x1 1G i 15nnHH 5 aE 11 mausm IgG x1 16nn 15HHnn 5 HgE 11 5 mause InG x1 1 15hnn 5 ngE 11 7 mouse InG x1100 15HHHH 5 09E 11 BB mouse InG 11 1 15nnnn 5 ngE 11 Mouse I G 1 15nnHH 5 aE 11 Sample Sample solution Flow rate Umin 5 ul min 100 l min 2 Mw Do D 20 C 20C Dilution Biacore T200 hethod Builder Variahles ssay steps Warlahl walues for assan Step Blark Blank Flow rate umin Sample Next Te CFCA Cycle run list Sample 1 MW
205. e l l I cnaLcuLaTE Diffusion coefficient qt 20 C b 03e ns l Convert diffusion coefficient from temperature T to 20 C Teinperatures T Ma temperature T 4 Ne 11 rts CONVERT hiffusian cnefficientnt 20 C 3 4de 11 mg 20C Molecular weight Da Friction ratio Choose molecular shaope lnhulaf 3 Globular 1 2 Moderately elongated 1 7 Elongated 2 5 Biacore 1200 4 95 Enter value Viscosity relative to water qt 20C 20C Use stqndard value 1 00 1
206. e series 4d Riemowe Selectior Znnm Inck Settings Single Sample series Biacore 1200 118 4 10 5 4 19 Close Next 4 19 Al sample series
207. ection Double Mix 96 well microplate 384 well microplate Biacore T200 158 7 1 7 1 1 Merged Inject Toolbar Run Wizard 2 Menubar Run gt Wizard OpenMew Wizard Template 5urface Preparation rmrnabilization pH Scouting son Melhos And Templates rmrnabilizatior ssau Development ETIml Buffer 5cnuhin EF GurfacE Peformance Hd17 I Lnntrnl Esnerimenls 1119 Kihetics Linked Reactiorns 2 Heneneralin Seouting 90521 Kinehics hass TrahsFET t Fietics a inity Bindinn nalysis Lnmcerilralinn nalusls Thgrmndpmamics ET Immunnnehnicih Immulmnnericitr Screenng Immunnnericltr LnnFrmatinn Immunanenicitu lntuhin Immunogenicity Immunogenicity Screening New Methods qnd Temploates Open
208. elative change 1 785 tc 7 016E 8 Rs IL mm Accept Curren t abort Curren Fitting Toble Fitting Accept Current Fitting Abort Current Abort Remaining Biacore 1200 40 2 Kinetics Affinity Kinetic Screen Fit Settings Taols Clear Flags Sample 2994 Liqand ref oxid Ritux Status IE Clea red Tools v Thumbnalls Hesults Summary Sensorgram View vy Arrange By Zoom lock GU 1000 1200 1400 1Bnn Display Blanks Display Blank Sublracted Samples Included Curyes In In D 0 D 0 In In D Result and Parameters Quality Control Report Residuals Parameters A a a es 8 6 Wm 1 1 Binding Quality Control Biacore T200
209. ent SAMPLE 3 I Regeneration lt REGENERATION 1 3 2 1 Biacore T200 4 1 Tre Binding Analysis Injection Parameters 5 ample Lnntact himg Flaw rale ymm Dissociation hmg Hanerigralinh Solutior 1Umh lp HCInHg High wiscosity solution Lnntact himg ls law rale lymiml Stabilization nerin mp Sample contact time s Flow rate umin Dissociation time s Regeneration Solution High viscosity solution 40 contact time s Flow rate l min Stabilization period Next es Binding Analysis Samnlps Sample table ample id 1 oi i mi Po PI i hir Sample id Control Ssqmples Biacore 1200 1 5
210. entration Analysis Enntrnl Samnles Controal sample defirition Hun control samples Repeat control samplels ewery sample cucles Control samples Expected conc Control sample id Control Sample definition Run control Samples Repeat control Sample s every 20 Control Samples Control Sample id Expected conc Next Concentratinn Analysis Samples Sample table Sample id Dilution factor 501 2435 01 501 2435 01 501 2436 01 501 2435 01 501 2435 02 S501 2435 02 501 2437 01 501 2437 01 501 2438 01 SH1 2438 1 501 2439 01 501 2439 01 S01 2440 01 501 2440 01 SOS 0001 01 S05 0001 01 TT Biacore T200 Sqmple Id Dilution factor Next EA A A EOPAVA Excel Excel txt
211. enubar File gt Save Evaluation Method As File gt Open File gt Apply Evaluation Method Biacore T200 3 53 3 kKinetics Affinity Biqcore T200 1 2 DMSO nM uM
212. erimndynamirs System Prenaratinns Frimg before run Mnrmalize detector I 1 Prime Normalize eThermodynamics Rack Positions Heaqent Hack 2 Type Sample 1 Sample 1 Conc nM Mw 1 118 antigen 4 118 antigen pr 11800 118 antigen Sample 11800 118 antigen Sample 11800 ee ee 118 antigen Sample 11800 118 antigen Sample 11800 118 antigen Sample 11800 XD XD 118 antigen Sample i 11800 118 antigen Sample 11800 dB well Microplate 118 antigen Sample 11800 118 antigen Sample 11800 118 antigen Sample 11800 a 1 909i0 10 118 antigen Sample 11800 I I I660 ee 118 antigen Sample 11800 B 118 antigen Sample 11800 i 118 antigen Sample 11800 118 antigen Sample 11800 118 antigen Sample 11800 118 antigen Sample 4 118nn 118 antigen Sample 11800 118 antigen Sample 11800 118 antigen Sample 3 5 11800 118 antigen Sample 17 11800 118 antigen Sample 11800 118 antigen Sample D 11800 118 antigen Sample 0 11800 118 antigen Sample 1 05 11800 118 antigen Sample 13 11800 118 antigen Sample 4 25 11800 1 ul
213. ethods aqnd Templqates Bia Users Don t Save Save in File name Save Standby flow Biacore T200 Evaluation Software Biacore 1200 164 7 7 2 Run Stop Run Biacore Tz This will stop the run Stop Run Run Stnhpe Finishin Current Cucle please wa bort cucle hn tlT Bre _ Ctrl Breakl
214. flow 6 2 Run Stop Run Biacore Tz A This will stop the run stop Run Run Stnhped Fimishimn Current cncle please mal bort cucle hn DTI Bre Ctrl Breakl Biacore T200 Evaluation Softwqre Biacore 1200 r RU OD d p 6 HI NN OO dU SE 3 dBHIIH Sh q HI DO DO 1II gi 3 II SH ELIH Td NN dh Timses 1000 RU 1 ng mmz 60 bound 0 recovered 4
215. had Builder 3 28 20HB Kinetics heteronenenus analyte kethod Builder 3 28 2HHB L1 lposnme capture Method Builder 3 28 20HB LMW kinetics Melhad Builder 3 28 2008 EE LM screen kethod Builder 3 28 20HB NTA kinetics Method Builder 3 28 20UB Single cycle kinetics kMelhod Builder 3 28 2UUB Calibration Free Concentration Open Method Builder Main Overview BiacoreT200 General Settings Biacore 1200 4 97 Ts Method Builder Main nveview 1 t star General Settings 2 Data collection rate Detection Sample compartment temperature 8 10 yl Hz Vary with analysis temperalure Assay Steps CcleTupes Maiable Selings 1 Miscellanenus 5 uffer settings Concentration unlt veiicaion_ gt lmesmenl me oo SepHun After run Specify an
216. ino 634 eamino 852 Damino 854 40 lt 0 8 3 sj amino 85a Cyeie Damino B34 Recos Port Adpustmert Rariong Ct ct Curve fttrg Carve Fumction Rhi Rio Al A2 che Ano OA CW Banienecul ocsnmds rameWtwr 3961 15 14 93 1177 0 70604 amino B34 aanino 3a Prameter 239 1 754 he 39 69 0 12035 ZAmino 83a 6034 Parmeter 33 1327 43 01 ID 0090s Zamino 8634 Geil neil sarede Perameter 33 49 190606 033 32 40 O135 Damino 63a Parmeter 399 0 19 tha3 30 01 5 2 Amino 3 rameWw 337 A093 5 15 3 0 ww 6O34 6O34 1 Lneer 4 oor Oon ylsesine Pinde PDMsclay Curre Paremeter y RByCokxz Cancel Report Point Table Evaluation Explorer Report Point Table Work areq Biacore T2nn Evaluation Software C MDocuments and Settings biacoreWDesktoptT100Manual2 CA_drug scr bme Report Point Table Fle View Evaluation Tools Windaw Help XX 3 a Sensorgram Plot BarChart gt Kinetics AFfinity gt Concentration Analysis lt Thermodynamics Ewaluation Explorer 1 Table Calumns x Remove window s AbsFesp HU
217. les are positioned by ordering the regions The first region in the list is positioned first Region Color Urientation Anchor Rack Yial Si i First Sort By Control sample cx an i Column Bottom leFt Sample Smal Ascending Sample EE CarkElue i Column Eottom left Sample Small Ascending Startup Crimson i Column Eottom left Sample Smal Ascending wash Oelow i Column Bottom left Reagent Large Ascending Salyent correction buffer a Elue i Column Bottom left Reagent Small Content Ascending gt Pooling Auto Poolingo_ Yes OK Automatic Positioning Biacore 1200 80 4 Eject Rack Roack tray port OK
218. lrmin prel RU s prel Ch 15HIH 1HIujml Prntein amp H5 H mouse InG 1T15 ni 15nn 5 15HHnn 1HHuniml Prntein a H5 H mause InG x1 OO 25 15IHHn anm lack B WE Flnww STing FInwer 1HTLIHmin 4 2 1 10 3 4 SI Timte CVCle Curve Ligand Sample Dilution factor Flow pl min Initial rate RU s OC ratio Temp MW Do D m2 s Blank used 7 5 12 5 5 oc Biacore 1200 116 4 Expand all cycles Use reference subtraction data
219. lumn Eottom left Sample Small Ascending Startup Crimson i Column Eottom left Sample Smal Ascending wash Oelow i Column Bottom left Reagent Large Ascending Salyent correction buffer a Elue i Column Bottom left Reagent Small Content Ascending gt Pooling Auto Poolingo_ Yes OK Automatic Positioning Biacore 1200 3 63 Eject Rack Rack tray port OK Eject Rack Tray Rqck Positions Next es drug Prepare Run Protocol Tahnma 10
220. manls Finetics Linked Haachinns Kinehics hass Trarnster EL Fietics a irnty Bindinn nalysis oneerntration nalusls Thgrmndprmamics EO Immunnnenicih Immulmnnerlcitr Screenng Immunnnemicltr LnnFrmatinn Immunanericltu lntuhin Screening Immunogenicity Immunogenicity Screening New Immunogenicity Screening Run Type To detect antibodies bound to drug in the sample samples may be acidified to release the antibodies and Ihen neutralized immediately before measurement How do you want to perform the measurements Direct analysis detects only free antibodies Acidification and neutralization detects antibodies in the presence of excess drug Direct analysis Merged Inject Next MI N Biacore 1200 194 7 Immunogenicity Screening Injectinn Sequence Detection Chip Flow path 13 Chip type ClM5 Liqand capture ArTDLE Sample Enhancement REGENERATIUN 1 Begeneraion lt Back New gt gese 1 Enhancement
221. meters Gample Contact kime 300 ls Flow rate plrmirn Mis snlulinri Frachinr Cucles mthnut drug Bufen mi snluhnn First reqerneratior Solutior High wiscosity solutior Contact hmg ls Flow rate 30 ymml 5CnTiH reqeneration olution High wiscosity solutior Contact hmg ls Flow rate ymm Stabilization period Etra wash after inectiorn with lsnnrnpahnHflaHH Sqmple Cycles without drug Buffer Cycles with drug Fraction Next Biacore T200 174 7 Immunogenicity Confirmatinn Samples Sample ahle Sample id Indiyid 3 Individ 3 Indiyid 13 Individ 13 Individ 1 Individ 1 Indiyid 10 Indiyid 10 Individ 7 Indiyid 7 Individ 18 Indiyid 18 Individ 2 Indiyid 2 Next Immunogenicity Confirmation ControlSamples Control sample definition Hun control samples Repeat control samplels every 10 sample cycles Control samples Control sample id
222. mmunogenicity lsotyping Cut off 2 2 Cut off Biacore 1200 7 193 CM 7 4 ADAs ADAs ADAs ADAs ADAs ADAs 7 4 1 Toolbar Run Wizard Menu bar Run gt Wizard Ww OpenMew Wizar Template 3uface Preparation Lnnk im Methoads nd Templates Immahillzahinn pH Enuhmn Immnhiizahinn Assar Deyelnnment Name Henengrahinh Seouting 030521 Bulfer Scouting na0723 5urface Performance EE Contol Exneri
223. mol kJzmagl EE Ema 45 as KTmmll 15 Tas t ass Fmol Finish Evaluation Explorer Thermodynamics Themodunamics ah eh Biacore 1200 6 1 2 ul 1
224. mp Sample 148 A amp nalyte amp Sample t hn CT lt Back Next gt Close Menu Automatic Positioning hutomatic Positioning Change the order in which the samples are positioned by ordering the regions The first region in the list is positioned first Region Color Urientation Anchor Rack Yial Si i First Sort By Control sample cx an i Column Bottom leFt Sample Smal Ascending Sample EE CarkElue i Column Eottom left Sample Small Ascending Startup Crimson i Column Eottom left Sample Smal Ascending wash Oelow i Column Bottom left Reagent Large Ascending Small Content Ascending Salyent correction buffer a Elue i Column Bottom left Reagent gt Pooling Auto Poolingo_ Yes OK Automatic Positioning Biacore 1200
225. ng s1 see below ntel Standhy after ruh wl use huffgr A hake sure there is ffash water in the water hnttle nn the right hand tray lf necessary Ermpty the waste bottle betore start of the run Estirmated run time bh Hmn enclading conditiornal staterments temperature charges ard starndby Ho Estimated butfer cornsumptior 2 Funninn bffer J atleast FUO ml plus 5H mydan for stahdhn after ruh Start Save qs Methods aqnd Templqates Bia Users Don t Save Save in File name Save Standby flow Biacore T200 Evaluation Software
226. nn the right hand tray e fnecessarY empty the waste bnttle befare start of the run Estimated run me 15h 32 min excludinn conditional statements temperature channes and standby flow Estimated bulfer consumptian Hunnina bulfer atleast 3HH ml plus 55 mlydaw for standhu after run Start Sqve qs Methods and Templqates Bia Users DontSave Save in File name Save Standby flow Biqcore T200 Evaluation Software Biacore 1200 188 7 7 3 1 Immunogenicity Isotybing
227. nogenicity Screening Immunagenicity Confirmation Immunogehnicity Isotyping Assay Thermodynamics New Methods and Templates Open Browse Open es Thermodynamics InjErtinn Sequence Detection Chip Flow path 1 Chip tnpe Capture REGENERATIN 1 Hegeneralion Ear ymr Le J ee 1 Biacore 1200 124 5 Detection Flow path 2 1 4 3 Chip Chip type Capture 2
228. nt Table Immunaqenicity onfirmation dE a Confirmation 2 Sensorgram Relative Respornse Tools Window Help XX Plot ui Bar Chart 3 Kinetics AFfinity gt Concentration Analysis gt Thermodynamics 4 Immunogenicity gt Settings Curve Fc 3 Report Point stability Sample cycles Mixed with drug Boundaries low lt 80 HU high gt 80 HU adiustments for controls No adjustments RU Sample Solution 240 a Individ 22 drug Individ 21 drug 3 Individ 20 drug Individ 3 drug Individ 13 drug 200 Individ 1 drug Individ 10 drug Individ 7 drug 180 Individ 18 drug Individ 2 drug 160 Individ 19 drug Individ 8 drug Individ 14 drug 140 Control Sample Individ 9 drug Mix salutian drug Individ 17 _dfug Individ 6 drug 120 _Individ 16 drug Individ 4 drug 100 i _Individ 11 drug Individ 15 drug Individ 12 drug 8n RA RK Individ 5 drug lt 128 3 6 9 9 Neg contral no drug 3 a Pos contral 1 pgiml no drug 60T Neq contral d KO i eg contro no drug Pos cantral 1 niml no drug 40 Neg control no drug 0 10 20 30 40 5 ED 70 Pos controlt1 Haiml no drug Cycle Humber Neg control no drug rann a i
229. ntroly Exactly 50 Startuptpos cnntral Exactly S50 Serum 3 Exactly S50 5erum 3 Exactly 5 5erum 3 Exactly 50 Serum 3 Exactly S50 5erum 3 i Exactly 50 Serum 3 i Exactly 50 Serum 3 i Exactly 50 Serum 3 Exactly 50 Serum 3 PP Exactly 5H Neg cnntral jml Control sample Control sample Control sample Control sample Control sample Control sample Control sample Control sample Control sample Control sample Control sample Control sample Control sample Control sample Control sample Control sample Startup Startup Startup Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample ww Exactly 50 Serum 3 ul Menu Export Positions
230. ol sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample Sample CT Sample 1 lt Back Next gt R1A12 12 Yial Si Small Rack Sample Sample Sample Reagent Reagent Anchor Eottom left Eottom left Eottom leFt Eottom left Eottom leFt Region Color Orientation First Sort By Ascending Ascending Ascending Ascending Content Ascending cyan EO CarkElue Startup A Crimsan wash Yelaw Salyent carrectinn buffer a Elue Control sample Sample E Column i Column Small E Small Large Small r E Column r Column s Column gt ge mw Er Biacore T200 8 1 Pooling Auto Pooling_ ves OK Automatic Positioning
231. ools ClearFlaas Sample 2994 Ligand ref oxidRitux Status E Cleared Tools Pumhnalls Hesults SunWW View vy Arrange By vr Sensorgram Zoom lock 2dd4 reF oxid Hitug BI Time Display Blanks Display Blank Subtracted Samples 1200 1400 Included Curyes View Small Thumbnails Standard Thumbnails Extended Thumbnails Arrqnge By Kinetic Screen Fit Settinns Taos ClearFlags Sample 2994 Ligand ref oxid Ritux Status Cleared Tools gt Thumbnails Hesults Summary Sensorgram View vy Arrange By vy Zoom lock 5 a 20 Display Blanks Display Blank Subtracted Samples 1400 Included Curves Ls J oe 36 bmp Paint Biacore 1200 34 2 Kinetics Affinity Display Blanks Inclu
232. ort Point Table Zoom Lock nl MT PT Cortrol Sample Sample Startup 1200 7 3 Keyword table Tools gt Keyword Table Edit Chip Information ee re bm ee e e EL e if se Ne we ee ee sr ss me se se ss EE ea ss Biacore T200 168 7 7 4 Menubor MM Toolbar am Curve Name Fc 1 44 Assay Step Purpose lt Dverlay gt Cycle lt Dverlay gt Sensorgram nd All sensorgrams RU Sensorgram Plot 75nnn ga Baseline Sample nr ga Binding stability H gi Controls enhancement 7oooo Se nso rg ram win d OW gi Controls stability ga Enhancement Peport Point Table BSnnn Hepart Point Tahle Evaluation Explorer og 1 SS000 Sample amp Startup Zoom Lock MMM MI Work area SOOO0 8nn 1000 1200 1400 1BQQ i s Menubar
233. ot settings Finish Biacore 1200 7 203 7 15 2 two component model 2 R Ri x e k97 t 10 R gt x e Kd2 t t0 R t 5 kaa kz
234. p cycles umber of cucles Startup Solution Number of cycles 3 Next gt Biacore T200 74 4 es Concentration Analysis Injection Parameters Sample Corntact him Flnw rate lmml Mim mlthi Frachinn of the mg snluhinn Hgngengrahinrn Solutior Fieg solution High wiscosity solution Corntact hm Sarmple Contact time Flow rate Mix with Regenerdtion Solution High viscosity solution contact time Flow rate Stabilization Deriod Next gt Biacore 1200 Flow rate lrmirnl Stabilization perind s s ul min Fraction 75 25 75
235. rder bs entered Increasing cnncehlralinn Sample id MW Do Concentration T nM ug ml Control Ssqmples Tes Kinetics hffinity Cnntrnl amples Control sample definitior Hun corntrol samples Fepeat control samplels yr sample cucles Lnnlrnl samples ww ba Concentration Concentration Control sample id Biacore T200 60 3 Control Sample definition Run control Sqmples Repeat control Sqmple s every Control Samples Next 3 3 Excel Excel Excel
236. rea Evaluation Explorer Biacore 1200 192 7 Toolbar Immunogenicty lt Isatyping UM Cuve EN gt Sample Cut RU Display isotyping reagent response as Hespanse J Response Rielative to Sample lsotyping Relative Response RU ABOB1 B23 B45SBEB7 HBCHC1C2C3C4C5S CECT CSCAIDODT D2DS DA4DSDEDT DD9ED E1 E2 E3E4 ESEG ET ES Sample Hame CUrVe Sample Cut Offt Cut Off 2 lgM lgG Cut Off Finish Biacore T200 Evaluation Software Ex5 lsotyping hlr lsotyping Fle Wiew Evaluation Tools Window Help x a 5ensoraram Plot al Bar Chart Kinetics AFfinity gt A Concentration Analysis Thermodynamics 3 Immunogenicity gt Curve Fc 2 Sample Cut Dff 290 RU Display isotyping reagent response as lsotyping Hepart Point Table mm Report Point Table I
237. s Calibration Tahle Calibration Curwe ps EE lt Eurye 1 Fitting Functior 4Parameter ng ml RU ng ml RU Biotin 1380 5 0 9948 16nn 1400 1200 1000 Relative Response co Lm mj mm Lm Parameters for curve 1 Rhi 93 52 Rlo 1432 Al 21 42 A2 1 051 Chr 13 43 40 5n 120 Concentration ndiTI Concentration Analysis Create Caqlibration Calibration Curve Settinds Flow cell Report point Response type Relative Response Slope Fitting Function linear 4 parameter Use Drecedind calibration Curve Use average calibration Curve Biacore 1200 Use calibration trends
238. stability gi Binding to reference Hepart Point Table 3nnnn mm Heport Point Table 35 In Conditioning Startup 1 25 nn Startuh 2 2 Startuh 3 Startuh 4 15 nn startup 5 Thermn 1 1 nnn Thermo 2 5nnn Therma 3 Thermo 4 Thermo 5 5 5 Keyword table Tools gt Keyword Table Edit Chip Information Ee ER ne EE ee ss ss se ee se se ee ee ss ss EE Biacore 1200 132 5 5 6 Menubor Toolbar Evaluation Explorer gt Work area Menubar Toolbar Evaluation Explorer ooram Pot Baseline Binding level Binding stability Binding to reference Work area Biacore T200
239. t Hg 1 J 1H 1HHI Tsd le5 5 Slow values above tt displayed E ITEdiuT high 1n ClE Humber mdium E DS medinm 5E Hz high 1 2E n3 medium E medium 1E 3 medium E Slow Fraction 7 15 RelResp Tot RU Fast Fraction Slow Fraction 9 t Fast s t1 2 Slow s Slow Fraction t 10 t Pl
240. tability Baseline RelResp Binding to reference Binding level Work qrea Evaluation Explorer Biacore 1200 Toolbar Immunogenicity stability Region settings Getthings Fiegions amp Boundaries umher of Fiegions La mm Lm Raative R es EE Fa Fa LT Lum LT Lm Lmm mm mm Settings CUTVe Report Point Response IVDe Regions amp Boundaries Number of Regions BoundQaryi Region Name Next 7 201 stabiity Response Tupe Helaliye Hgspnrse Fiegion ame Bourndar high 3HH mdium 11 CwrlE Humber stability Relative Response Biacore T200 202 7 Stahility Gelect data medium 150 300 RU high ELH RU hiahne plo
241. ty Wizard Biacore T200 6 Biacore T200 66 3 3 2 Evaluation Biacore T200
242. ues in method Deline some yalues in method and others at run time Cycle Tupes Variable Settings alues for these variables will be defined at run time Command variable Dt20 C Elank SeupRun Diution Assay Steps Blank Define some values in Method and others at run time Flow rate ul min Setup Run Sample solution Buffer MW Do D 20 Blank Yes _ Dilution Verification Biacore 1200 4 105 Ts Method Builder Main yeryiew Verlficalion results The method has been verified and can be used to set up a run Assay Steps Cycle Tupes Wariable Settings vericaion The Method has been verified qnd can be used to set up a run Setup Run Rethod Buil
243. ution 40 contqct time 60 s Flow rate 30 ul min Stqbilization period 0s Next hernmadynamics Samples Samples 5arnnle id ww hai I HHH D OO 1g51 ig513 II5H15 1 3 ZD 1g51 Hun nrder LAs entered Increasing cnncehlralinn Sample id MW Do Concentration nM ug ml I Next 5 1 Excel Excel Excel txt Biacore T200 5 127 Th
244. ve Result Plnt Curve Name Fc 2 1 carr Assay Step Furpose lt Hyerlaw gt rj Cycle lt Hverlay gt rj Result Plot Zoom Lock 5 3 H69 negative 7 93 22 STOSDZ 8 9 0 4068 T_5 2233 T_6 12 5 17 2 13 2 16 3 14 5 569 ED Corntrol sample 16 HH1698 5 17 H 2355 7 Sample 18 3 528 negative pg E gE 1 5 s 4 20 19 3 528 negative 2 97 75 STOSDZ 21 2 3 7 A 8 22 24 15 A 9 23 111 2 1 24 1 69 B_1 25 1862 B_2 26 4 346 B_3 Heport Point Adiustment Hanking Cutoff Curve fitting i NR 5 28 5335 BE_5 y Molecular weight amp djustment 29 10 55 B_6 3H 1 7Z24 B_ Adjustment For Controls Molecular weight Adiustment Positive Control name STO802 Negative Control name negative Capture Adjustment Fitting Function Linear 31 5 1HG negative 32 5 1HG negative a 33 94 5 STOSDZ adjustment For Controls y Median Filtering a Upper range limit 10 34 16 2 BE 8 Lower range limit 5 35 ni6171 B 9 kMedian Filterina indawi width 10 v 36 3 7B6 B_10 Ranking Cut off Ranking Cut off Ranking Cut off Biacore 1200 24 1

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