CHO Host Cell Protein Detection

Transcription

1 TECHNICAL NOTE 41 CHO Host Cell Protein Detection TABLE OF CONTENTS OVERVIEW... 1 PRINCIPLE... 2 ASSAY ACCURACY AND SPECIFICITY... 2 Mterils Required...2 STORAGE AND STABILITY... 3 ASSAY MATRIX... 3 OCTET HTX SYSTEM HCP ASSAY PROTOCOL: A COMPLETELY WALK-AWAY ASSAY... 3 Prepre Smples nd Detection Pltes...3 Run the Assy...4 Anlyze Dt...4 Exmple Dt from Routine Assy...5 OCTET QK384, QK E, RED384, AND RED96 SYSTEM HCP ASSAY PROTOCOL WITH SIDEKICK STATION... 5 Process Smples on the Sidekick Sttion...5 Run the Assy...7 Anlyze Dt...7 Exmple Dt from Routine Assy....7 OVERVIEW Host cell proteins (HCPs) re contminnts found in biophrmceuticls expressed in bcteril, yest or mmmlin production cell lines. Among protein expression cell lines, Chinese hmster ovry (CHO) cells re the most commonly used mmmlin hosts for industril production of recombinnt protein therpeutics. However, mnufcturing nd production processes of biophrmceuticls often leve behind contminting HCPs from CHO cells. Such residul HCPs crry substntil risk of decresing efficcy of the drug nd cusing dverse immunogenic rections in ptients. Hence, the detection of residul host cell protein contminnts nd methods tht reduce them to the lowest cceptble levels hve become criticl spects of drug sfety nd qulifiction. HCP nlyses cn be brodly clssified into two ctegories, generic ssys nd process-specific ssys. Commercilly vilble generic ssys re intended to detect HCPs tht might contminte product independent of downstrem purifiction processes. These ssys normlly use HCPs obtined from fr upstrem (e.g., conditioned medi) or fter some minimlly selective purifiction step (e.g., clrifiction or filtrtion) s n ntigen to generte brodly-rective polyclonl ntibodies for detection. They re very useful when most HCPs re conserved mong relted strins nd processes. On the other hnd, process-specific ssys re those derived from n immunogen which is specific to defined purifiction process. These ssys re tilor-mde for n estblished process nd thus hve the potentil to be more specific to the pnel of HCPs produced within such processes. However, high specificity for typicl HCPs occurring within the process crries the risk tht ny typicl HCPs rising due to unintended nd undetected devitions in purifiction process my go undetected potentilly leding to substntil setbcks in the drug development timeline. 1 The vst mjority of process-specific ssys re developed only fter process is set nd defined, which typiclly occurs t the lter stges of drug development. Detection ntibodies for processspecific ssys lso require substntil time nd cost to develop. Since the FDA generlly does not require process-specific HCP nlyses until phse III trils, generic ssys re typiclly employed for overll HCP detection until drug cndidte survives the initil

2 Anti-CHO HCP Biosensor Cygnus 3G nti-hcp up to run utomticlly on n Octet HTX instrument with results obtined in one hour. The ssy cn lso be run on other 8- nd 16-chnnel Octet instruments together with the Sidekick Sttion with time-to-results of 75 nd 90 minutes, respectively. Precipittion phses of clinicl trils. Even fter process-specific ssy is developed nd implemented, generic ssys cn still be used routinely to complement process-specific testing to mximize detection of ny potentil typicl HCPs. Becuse of ll these fctors, generic ssys will lwys remin the most esily ssessble nd highly useful tools in ny drug development progrm. PRINCIPLE HRP HRP HRP HRP Figure 1: Biosensor-bsed ssy formt for the detection of CHO HCP. Among existing HCP nlyticl methods, ELISA is perhps the more commonly used nlyticl method. Western blots nd SDS-PAGE re lso used, but re limited by their qulittive nture nd lck of quntittion sensitivity. There re severl inherent problems with ELISA, stemming from its relince on highly mnul processing steps tht introduce vribility in mesurement, multiple time-consuming incubtion steps, nd relince on colorimetric or fluorescent probes tht cn yield flse positive signls. Pll ForteBio s Octet pltform provides superior lterntive to ELISA with improved precision in mesurements, better or equivlent sensitivity nd dynmic rnge, low user intervention, rpid ssy development enbled by rel-time monitoring, nd much fster time-to-results. The pltform is used for generic CHO HCP ssys in erly phses of clinicl product development, nd process-specific CHO HCP ssys cn lso be constructed using the sme ssy formt if process-specific ntibody hs been developed. This Technicl Note outlines protocol for using the Pll ForteBio- Cygnus Anti-CHO HCP Detection Kit in developing nd routine running of process-independent ssys on the Octet pltform. The mesurement involves sndwich-type ssy on n Anti-CHO HCP Biosensor tht is pre-coted with the gold-stndrd 3G Anti-CHO HCP ntibody from Cygnus Technologies (Figure 1). A completely hnds-off, wlk-wy HCP ssy nlyzing 96 smples cn be set HRP HCP Fluorescein-tgged nti-hcp HRP nti-tg conjugt Metl DAB ASSAY ACCURACY AND SPECIFICITY In certin cses, the drug products themselves or components in the formultion buffer my interfere with the ssy s bility to detect HCPs. Fctors such s extremes in ph, detergents, orgnic solvents, high protein concentrtion, nd high buffer slt concentrtions re ll potentil interference fctors. It is therefore necessry to vlidte by estblished experimentl procedures (i.e. ICH nd FDA guidelines) tht the ssy results will be ccurte. We recommend users perform two criticl experiments in estblishing ssy ccurcy nd specificity: spike recovery nd dilutionl linerity (plese lso refer to ASSAY MATRIX on pge 3). If it is determined tht there is significnt product or mtrix interference in the ssy, further dilution or buffer exchnge of the product to render it into more ssy comptible buffer might be necessry. The sme diluent used to prepre the kit stndrds is idelly the preferred mteril for dilution or buffer exchnge of your smples. For ech smple type to be tested, users should demonstrte tht the ssy cn recover dded HCP or other contminnts spiked into tht smple mtrix. This cn be performed by spiking the highest stndrd provided with the kit into your smple types nd then testing in the ssy. MATERIALS REQUIRED Octet HTX, QK384, QK e, RED384, or RED96 instrument with Octet Dt Acquisition nd Anlysis Softwre version 8.1 or lter. Sidekick Offline Biosensor Immobiliztion Sttion for high throughput ssys if using Octet QK e, QK384, RED96, or RED384 instrument (not needed with the Octet HTX instrument). Blck polypropylene 96-well or 384-well micropltes (Greiner Bio-One prt no or ). Volume of smples to be nlyzed (including positive nd negtive controls): 80 µl (384-well microplte) or 200 µl (96-well microplte). Anti-CHO HCP Detection Kit (Pll ForteBio LLC prt no ), contining the following: Anti-CHO HCP Regents A CHO Antigen, 48 µl, 20 µg/ml Fluorescein nti-cho, 240 µl, 100X concentrte Anti-FITC HRP, 480 µl, 50X concentrte Smple Diluent with Proclin 300, zide-free, 3 x 50 ml Anti-CHO HCP Regents B Metl Enhnced DAB concentrte, 2.4 ml, 10X concentrte Stble Peroxide Buffer, 46 ml One try of 96 Anti-CHO HCP biosensors 2

3 STORAGE AND STABILITY Anti-CHO HCP Regents A should be stored t 4 C. The CHO Antigen cn be stored short-term up to one month t 4 C nd long-term t -20 C. More thn three freeze-thw cycles should be voided. Anti-CHO HCP Regents B should be stored t -20 C. Stble Peroxide Buffer cn be stored t 4 C fter first use upon thwing. Metl Enhnced DAB should lwys be stored t -20 C. Anti-CHO HCP biosensors should be stored t room temperture. ASSAY MATRIX Differences between mtrices cn potentilly influence ssy performnce. Diluting the smple mtrix using Pll ForteBio s Smple Diluent with Proclin 300 is n effective wy to minimize mtrix effects. Prior to running the ssy, we recommend n optimiztion step where smples re diluted with vrying mounts of Smple Diluent in order to determine the minimum dilution fctor required for optiml ssy performnce. It is lso importnt to test the dilution linerity of the smple to ensure no interference from mtrix. OCTET HTX SYSTEM HCP ASSAY PROTOCOL: A COMPLETELY WALK-AWAY ASSAY The entire CHO HCP ssy cn be set up on the Octet HTX system to enble high throughput, wlk-wy ssys. This utomted ssy formt elimintes ny potentil user-relted dt vrition tht cn be cused by mnul processing nd helps chieve the most stremlined nd efficient lbortory workflow. A full ssy cn be performed nd dt obtined in bout one hour with excellent ssy precision nd run-to-run consistency. Prepre Smples nd Detection Pltes NOTES: All buffers nd diluents used in this ssy should be zide-free. The presence of zide cn inhibit the ctivity of the HRP enzyme. Protect Fluorescein nti-cho, Anti-FITC HRP, nd Metl Enhnced DAB regents from light. Plese refer to the mterils sfety dt sheet (MSDS) for sfety informtion on the Metl Enhnced DAB concentrte. Dispose of unused nd used regent in ccordnce with ll locl, stte, nd federl guidelines. Proper personl sfety mesures should lso be tken when hndling hzrdous mterils. 1 Equilibrte the smples, regents nd buffers to room temperture nd mix thoroughly prior to use. Metl Enhnced DAB concentrte should remin t -20 C until immeditely before use. 2 Prepre ech concentrtion of HCP clibrtion stndrd in Smple Diluent with Proclin 300. The clibrtion stndrds selected should cover the HCP ssy rnge 3 from ng/ml, nd the recommended clibrtor concentrtions re 0.5, 1, 2, 8, 25, 75, nd 200 ng/ml. It is lso recommended to run buffer-only controls s references. A clibrtion curve should be included in ech run. 3 Prepre the Smple Plte (exmple plte lyout shown in Figure 2): Pipette 80 µl of stndrd nd unknown smples prepred in Smple Diluent with Proclin 300 into the wells of 384- well microplte. b Pipette 80 µl of Fluorescein nti-cho prepred t 1:100 dilution in Smple Diluent with Proclin 300 into the wells of the 384-well microplte. c 1 A B C D E F G H I J K L M N O P Pipette 80 µl of Smple Diluent with Proclin 300 into the wells of the 384-well microplte Stndrds nd smples prepred in Smple Diluent with Proclin 300 Fluorescein nti-cho prepred 1:100 in Smple Diluent with Proclin 300 Buffer: Smple Diluent with Proclin 300 Figure 2: Smple Plte lyout for 96 biosensor mode in 384-well plte for Octet HTX system A B C D E F G H I J K L M N O P Enzyme: Anti-FITC HRP 1:50 in Smple Diluent with Proclin 300 Detection regent: Metl DAB 1:10 in Stble Peroxide Buffer 2nd Buffer: Stble Peroxide Buffer Unused wells Figure 3: Detection Plte lyout for 96 biosensor mode for Octet HTX system.

4 Figure 4: Settings for Advnced Quntittion ssys for HCP detection on the Octet HTX system. The left pnel shows the Smple Plte configurtion nd ssy settings in 96 biosensor mode which contins smple incubtion step, n Fluorescein nti-cho incubtion step, nd two wsh steps with Smple Diluent. The right pnel shows the Detection Plte configurtion nd ssy settings for detection using 96 biosensor mode. 4 Prepre the Detection Plte (exmple plte lyout shown in Figure 3): b c Pipette 80 µl of Anti-FITC HRP prepred t 1:50 dilution in Smple Diluent with Proclin 300 into the wells of 384-well microplte. Pipette 80 µl of Stble Peroxide Buffer into the wells of the 384-well microplte. Pipette 80 µl of Metl Enhnced DAB prepred t 1:10 dilution in Stble Peroxide Buffer into the wells of the 384-well microplte. 5 Prepre the Biosensor Try. Biosensor loctions should correspond to filled wells in the Smple Plte (one biosensor for ech filled well in the Smple Plte). 6 Prepre Hydrtion Plte by pipetting 200 µl of Smple Diluent with Proclin 300 into ech well of 96-well polypropylene plte. Well loctions filled with Smple Diluent in the Hydrtion Plte should correspond to biosensor loctions in Biosensor Try. Run the Assy 1 Plce the Smple nd Detection Pltes in the Octet HTX instrument on the defined plte sttions. 2 Lunch Octet Dt Acquisition Softwre nd choose the Advnced Quntittion option in the Experiment Wizrd. In the Assy Settings window, click Modify to set up the ssy prmeters s shown in Figure 4. The 96 biosensor mode settings re shown in the left nd right pnels. Note tht the long smple incubtion nd Fluorescein nti-cho incubtion steps (run in 96 biosensor mode) re defined in the Sensor Loding tb, nd the remining steps re defined in the Plte Definition tb. Alterntively, users with Octet Dt Acquisition Softwre v cn open pre-set method files by choosing Experiment from the min menu, then Templtes, then Quntittion, 4

5 then Advnced Quntittion, nd choose the pproprite method file. 3 Click OK when ssy prmeters hve been defined. 4 Define the Smple Plte lyout to correspond to the lyout in Figure 2. 5 Define the Detection Plte lyout to correspond to the lyout in Figure 3. 6 Enter Smple nd Sensor informtion in the Plte Definition tb nd the Sensor Assignment tb s desired. 7 In the Run Experiment tb, enter dely time of 600 seconds in order to give the pltes t lest 10 minutes inside the Octet instrument to equilibrte to ssy temperture. 8 Enter loction nd file nme for sving the dt. 9 Click GO to run the ssy. Anlyze Dt The nlysis of the dt obtined on the Octet HTX system is identicl to the nlysis of dt obtined from other instruments. To nlyze the dt: 1 In Octet Dt Anlysis Softwre, lod the dt folder to be nlyzed. 2 Select the reference well nd perform reference subtrction if needed. 3 Group nd Concentrtion informtion cn be modified in the tble if needed. 4 In the Results tb: Select R equilibrium s the binding rte eqution. This eqution will fit the binding curve generted during the experiment nd clculte response t equilibrium s the output signl. b Click Clculte Binding Rte. Results will be displyed utomticlly in the tble. c Click Sve Report or select File > Sve Report to generte Microsoft Excel report file. Exmple Dt from Routine Assy Binding (nm) Time (sec) Expected Concentrtion (ng/ml) Clculted Concentrtion (ng/ml) Recovery %CV % 4.2% % 5.0% % 4.4% % 6.2% % 4.2% % 5.0% % 9.8% Figure 5: Exmple dt from CHO HCP ssy on the Octet HTX system. The dt shows dose response for the clibrtion stndrds (N=12). The clculted concentrtions nd %CV vlues resulting from the nlysis of the dt re shown in the ccompnying tble. OCTET QK384, QK E, RED384, AND RED96 SYSTEM HCP ASSAY PROTOCOL WITH SIDEKICK STATION The HCP ssy for 8- nd 16-chnnel Octet instruments is performed ccording to the steps shown in Figure 6. When using these systems, the initil incubtion steps re processed using the Sidekick Sttion. After biosensors re incubted with HCP smples nd Fluorescein nti-cho ntibody, the remining steps re performed on the Octet instrument. Process Smples on the Sidekick Sttion NOTES: All buffers nd diluents used in this ssy should be zide-free. The presence of zide cn inhibit the ctivity of the HRP enzyme. Protect Fluorescein nti-cho, Anti-FITC HRP, nd Metl Enhnced DAB regents from light. 5

6 Sidekick Sttion: Hydrtion Plte (200 µl/well) Smple Diluent Proclin min, 30 C, 1000 rpm Sidekick Sttion: Wsh Plte (200 µl/well) Smple Diluent Proclin secs, 30 C, 1000 rpm Sidekick Sttion: CHO HCP Plte (200 µl/well) CHO HCP Stndrds nd Smples 30 mins, 30 C, 1000 rpm Sidekick Sttion: Fluorescein nti-cho Plte (200 µl/well) Fluorescein nti-cho ntibody 30 mins, 30 C, 1000 rpm with Smple Diluent in the Hydrtion Plte should correspond to biosensor loctions in Biosensor Try. 6 Using the Sidekick Sttion, hydrte biosensors in the Hydrtion Plte t 1000 rpm nd 30 C for 1 minute. 7 Incubte hydrted biosensors in the Smple Plte on the Sidekick Sttion t 1000 rpm nd 30 C for 30 minutes. 8 During this incubtion, prepre the Fluorescein nti-cho Antibody Plte: Prepre 1:100 dilution of Fluorescein nti-cho ntibody in Smple Diluent with Proclin 300. Sidekick Sttion: Wsh/Hydrtion Plte (200 µl/well) Smple Diluent Proclin secs, 30 C, 1000 rpm Octet System: 600 sec, RT, 0 rpm Plese refer to the sfety dt sheet for sfety informtion on the Metl Enhnced DAB concentrte. Dispose of unused nd used regent in ccordnce with ll locl, stte, nd federl guidelines. Proper personl sfety mesures should lso be tken when hndling hzrdous mterils. 1 Equilibrte the smples, regents nd buffers to room temperture nd mix thoroughly prior to use. Metl Enhnced DAB concentrte should remin t -20 C until immeditely before use. 2 Prepre ech concentrtion of HCP clibrtion stndrd in Smple Diluent with Proclin 300. The clibrtion stndrds selected should cover the HCP ssy rnge from ng/ml, nd the recommended clibrtor concentrtions re 0.5, 1, 2, 8, 25, 75, nd 200 ng/ml. It is lso recommended to run buffer-only controls s references. A clibrtion curve should be included in ech run. 3 Prepre the Smple Plte (exmple plte lyout shown in Figure 7): b Octet System: Detection Plte (200 µl/well) Buffer (Smple Diluent Proclin 300) Enzyme (Anti-FITC HRP ntibody) 2nd Buffer (Stble Peroxide Buffer) Detection (Metl DAB) Figure 6: Flow chrt of HCP ssy steps on 8- nd 16-chnnel Octet systems with the Sidekick Offline Immobiliztion Sttion. Pipette 200 µl of ech stndrd into the wells of 96-well microplte. Pipette 200 µl of ech unknown smple into the wells in the reminder of the microplte. 4 Prepre the Biosensor Try. Biosensor loctions should correspond to filled wells in the Smple Plte (one biosensor for ech filled well in the Smple Plte). 5 Prepre one Hydrtion Plte nd two dditionl Wsh Pltes by pipetting 200 µl of Smple Diluent with Proclin 300 into ech well of three polypropylene 96-well pltes. Well loctions filled 6 b Pipette 200 µl into ech well of new 96-well microplte. Filled wells should correspond to biosensor loction nd number in the Biosensor Try. 9 After 30 minutes of smple incubtion, replce the Smple Plte with the first Wsh Plte on the Sidekick Sttion nd incubte t 1000 rpm nd 30 C for 30 seconds. 10 Replce the Wsh Plte with the Fluorescein nti-cho Antibody Plte. Incubte on the Sidekick Sttion t 1000 rpm nd 30 C for 30 minutes. 11 During this incubtion, prepre the Detection Plte. Either 96-well or 384-well microplte cn be used depending on the Octet instrument being used. The fill volumes for 96- nd 384- well micropltes re 200 µl nd 80 µl, respectively. b Prepre n pproprite volume for the plte type of: A 1:50 dilution of the Anti-FITC HRP ntibody in Smple Diluent with Proclin 300 A 1:10 dilution of Metl Enhnced DAB in Stble Peroxide Buffer In blck, flt-bottomed 96- or 384-well microplte, pipette 200 µl or 80 µl respectively of ech regent specified into the wells of the microplte using the plte lyout shown in Figure 8). Ech regent needs to be filled into ll wells of one column (for 8-chnnel ssys) or two columns (for 16-chnnel ssys) in the Detection Plte. 12 After 30 minutes of Fluorescein nti-cho incubtion, replce the Smple Plte with second Wsh Plte on the Sidekick Sttion nd incubte t 1000 rpm nd 30 C for 30 seconds. Run the Assy 1 After the second wsh step on the Sidekick Sttion is complete, plce the Biosensor Try over the second Wsh Plte (the sme plte used on the Sidekick Sttion for the lst wsh) into the Octet instrument. 2 Plce the Detection Plte into the Octet Instrument. For Octet QK e, RED, nd RED96 systems, plce the Detection Plte in the single plte sttion (Plte 1) in the instrument. For Octet RED384 nd QK384 systems, plce the Detection Plte in the regent plte sttion (Plte 2).

7 A B C D E F G H Figure 7: Smple Plte lyout for incubtion with biosensors on the Sidekick Sttion. A B C 1 HCP stndrds HCP unknown Figure 9: Settings for Advnced Quntittion ssys for HCP detection on 8- nd 16-chnnel Octet systems. 3 Lunch Octet Dt Acquisition Softwre nd choose the Advnced Quntittion option in the Experiment wizrd. In the Assy Settings window, click Modify to set up the ssy prmeters s shown in Figure 9. Note tht the smple step is specified to hve been crried out offline. 4 Click OK once the ssy prmeters hve been defined. 5 Define the Smple nd Detection Plte lyouts: For Octet RED384 nd QK384 systems: D E F G H Define the Smple Plte lyout to correspond to the lyout in Figure 7. The Smple Plte Lyout needs to be defined in the Octet Dt Acquisition Softwre even though the Smple step hs been performed offline s this enbles the softwre to ssign the correct Smple IDs to the biosensors during nlysis. 1 A B C D E F G H I J K L M N O P Smple Diluent Anti-FITC HRP Stble Peroxide Buffer b Define the Detection Plte lyout to correspond to the pproprite lyout in Figure 9: Smple Diluent with Proclin 300 = B (Buffer) Anti-FITC HRP ntibody = E (Enzyme) Stble Peroxide Buffer = 2 (2nd Buffer) Metl Enhnced DAB = D (Detection) For Octet RED96 nd QK e systems: Specifying the Smple step to be crried out offline enbles two pltes to be opened in the softwre (even though there is only one plte position in the instrument). The first plte (Plte 1) hs the sme lyout s the Smple Plte lyout used in the Sidekick Sttion incubtion. Figure 8: Detection Plte lyout for 8-chnnel ssys on Octet RED, RED96 nd QK e systems (top) nd for 16-chnnel ssys on Octet RED384 nd QK384 systems (bottom). 7

8 b The lyout of the second plte (Plte 2) is defined to correspond to the pproprite Detection Plte lyout in Figure 9 where: Smple Diluent in Proclin 300 = B (Buffer) Anti-FITC HRP ntibody = E (Enzyme) Stble Peroxide Buffer = 2 (2nd Buffer) Metl Enhnced DAB = D (Detection) 6 Enter Smple nd Sensor informtion in the Plte Definition tb nd the Sensor Assignment tb s desired. 7 In the Run Experiment tb, enter dely time of 600 seconds in order to give the pltes t lest 10 minutes inside the Octet instrument to equilibrte to ssy temperture. 8 Enter loction nd file nme for sving the dt. 9 Click GO to run the ssy. Anlyze Dt 1 In Octet Dt Anlysis Softwre, lod the dt folder to be nlyzed. 2 Select the reference well nd perform reference subtrction if needed. Binding (nm) Time (sec) Figure 11: Rel time dt from detection steps mesured on the Octet RED384 system with the CHO HCP ssy. Processing of the biosensors with smples nd the binding of the Fluorescein nti-cho ntibody were performed using the Sidekick Sttion. The steps shown include 1) bseline in Smple Diluent, 2) binding of Anti-FITC HRP ntibody, 3) Stble Peroxide Buffer equilibrtion nd 4) detection of signl using Metl Enhnced DAB substrte. The ssy ws run ccording to the procedure outlined in this technicl note Group nd Concentrtion informtion cn be modified in the tble if needed. 4 In the Results tb: Select R equilibrium s the binding rte eqution. This eqution will fit the binding curve generted during the experiment nd clculte response t equilibrium s the output signl. b Click Clculte Binding Rte. Results will be displyed utomticlly in the tble. c Click Sve Report or select File > Sve Report to generte Microsoft Excel report file. Binding (nm) Time (sec) Exmple Dt from Routine Assy The dt shown ws generted on the Octet RED384 system using the protocol outlined in this technicl note. CHO-HCP stndrds t 200, 75, 25, 8, 2, 1, nd 0.5 ng/ml in Smple Diluent with Proclin 300 were prepred nd run in triplicte. Three unknowns (Smples 1, 2 nd 3) were run in 8 replictes to ssess ssy precision. Expected Concentrtion (ng/ml) Clc. Concentrtion (ng/ml) Recovery %CV % 2.1% % 0.7% % 2.3% % 2.8% % 1.8% % 1.7% % 3.3% Figure 10: The dt shows dose response for the clibrtion stndrds in triplicte. The clculted concentrtions nd %CV vlues resulting from the nlysis of the dt re shown in the ccompnying tble. 8

9 Binding (nm) Smple Clc. Concentrtion (ng/ml) %CV Smple % Smple % Time (sec) Smple % Figure 12: Grph showing dt for three unknown smples, ech hving eight replictes. The clculted concentrtions nd %CV re shown in the ccompnying tble. The concentrtions were clculted using the clibrtion dt shown in Figure 11. ASSAY TROUBLESHOOTING GUIDE Issue Possible Resons Suggested Solutions Stble Peroxide Buffer nd Smple Diluent switched. Re-run ssy tking cre to dilute the Metl Enhnced DAB concentrte in Stble Peroxide Buffer nd smples nd stndrds in Smple Diluent with Proclin 300. No signl from HCP stndrds Stble Peroxide Buffer or Metl Enhnced DAB is no longer ctive. Check for ctivity by combining Stble Peroxide Buffer nd Metl Enhnced DAB with 1 μl of HRP nti-fitc ntibody. A drk precipitte should pper if ll re ctive. Regents no longer ctive. Confirm tht the Metl Enhnced DAB hs been stored t -20 C, the Stble Peroxide Buffer t either -20 C or 4 C (fter opening), the biosensors t room temperture nd the CHO stndrds, Fluorescein nti-cho nd HRP nti-fitc t 4 C. No smple signl HCP concentrtion is below the LOD. Perform dilution series of the smple in order to determine wht dilution is required to bring the smple signl within the dynmic rnge of the ssy. Azide present in smples. Dilyze smple to remove zide. 9

10 Issue Possible Resons Suggested Solutions Smple signl is too high HCP concentrtion is bove the concentrtion of the highest stndrd. Perform dilution series of the smple in order to determine wht dilution is required to bring the smple signl within the dynmic rnge of the ssy. Inconsistency in incubtion times. For best results, ssy step times should be consistent cross stndrds nd smples. For incubtion steps involving mnul intervention, use lb timer to ensure ll smples nd ssys re treted similrly. High vribility between runs Fluorescein nti-cho, nti-fitc HRP nd Metl Enhnced DAB not protected from light. Protect Fluorescein nti-cho, nti-fitc HRP nd Metl Enhnced DAB from light by storing in drk, dry plce when not in use. Additionlly, dilutions of these mterils should be stored in drk plce until they re pipetted into micro pltes nd plced either into the instrument or onto the Sidekick Sttion. Pll ForteBio Corp Willow Rod, Suite 201 Menlo Prk, CA t: 888.OCTET-75 or Pll ForteBio Europe 5 Hrbourgte Business Prk Southmpton Rod Portsmouth, PO6 4BQ, UK Pll ForteBio Anlytics (Shnghi) Co., Ltd. No. 88 Shng Ke Rod Zhngjing Hi-tech Prk Shnghi, Chin t: , Pll Corportion. Pll,, ForteBio, Octet, BLItz, Dip nd Red, Suprcp nd Seitz re trdemrks of Pll Corportion. indictes trdemrk registered in the USA nd indictes common lw trdemrk. TN-4041 Rev A