L3 team Update September 10, 2012

Transcription

1 L3 team Update September 10, 2012 Summary: Overview of team Suggested best practices for the platform criteria Platform review of specific criteria and pros and cons of each All comments and opinions to be sent to

2 L3: Assay formats Team members: Team lead Sherri Dudal EU Other members Alison Joyce NA Rebecca Crisino NA Jihong Yang NA Yoshitaka Taniguchi APAC Karolina Osterlund EU John Smeraglia EU Katherine McKay EU Jaya Goyal NA Daniel Baltrukonis NA Mahesh Kumar APAC (stopped activity) In scope Assay platforms for LBAs Gyros, MSD, Biacore, AlphaLISA, Delfia, Singulex, Luminex, Immuno-PCR, Cell-based assays, RIA Acceptance criteria for these methods for both validation and sample analysis How to set up the assays placement of standards and QCs in these new formats Pros and cons of using these formats Multiplexing with these formats and criteria required Interdependencies with other teams L1 Large molecule specific run acceptance: acceptance criteria for new methods/platforms versus ELISA 96 well plate Out of scope L2: set-up of a balanced design for 96 well ELISA L4: stability of critical reagents L5: any automation activities linked to the platform 2

3 Strategy The guidelines and practices described by FDA, EMA, JBF, APAC were combined into one working document. China follows the guidelines that are currently in place so no adjustment was needed. The ANVISA guidelines are very small-molecule oriented and thus do not differ from current recommendations. The team was divided into work groups to focus on the different platforms and bring information and discussion points to the overall team. Each team has been formed to ease time differences and is grouped according to expertise with a particular platform. The platform issues, criteria and pros and cons have been presented and discussed within the team and these will now be presented to colleagues at the workplace and in forum discussion groups to obtain more feedback. The work groups were organized as follows: Platform Leader Team member Team member Gyros Karolina (EU) Sherri (EU) Alison (NA-E) Cell-based assays Daniel (NA-E) Yoshitaka (APAC-Japan) Jaya (NA-E) RIA Mahesh (APAC-India) Daniel (NA-E) 384-well format John (EU) Karolina (EU) Alpha-ELISA/Delfia Rebecca (NA-E) Jaya (NA-E) John (EU) Singulex Alison (NA-E) Rebecca (NA-E) Mahesh (APAC-India) Biacore Sherri (EU) Jihong (NA-W) Alison (NA-E) MSD multiplex Katherine (EU) Yoshitaka (APAC-Japan) Karolina (EU) Luminex multiplex Jihong (NA-W) Katherine (EU) Jaya (NA-E) Immuno-PCR Jaya (NA-E) Jihong (NA-W)

4 Accomplishments Our team kicked off our first meeting on July 26 th and since then, we have been working hard on discussing and coming up with criteria for the different platforms. This has involved a lot of commitment from all and it is the moment to highlight our accomplishments over the last year. We have discussed, sorted, critiqued, and reviewed a number of aspects of which the following cover the general themes: Individual platform descriptions Individual platform pros and cons Individual platform acceptance criteria comparisons Criteria for multiplexing: Luminex and MSD Classification of platforms in series: Gyros, Erenna, Biacore (Ria would follow the same) EMA, FDA, JBF guideline comparisons -- little differences outside of EMA and FDA Compilation of criteria for platform comparison Slide deck for presentation at WRIB, NBC, EBF, and JBF All the feedback and input during the meetings!

5 Seeking further feedback We are now at the point of seeking feedback to make our recommendations or suggested best practices global. For this, we require further feedback for: Sample analysis in series (calibrators and quality control placement)? When can singlet analysis be put in place Multiplexing? Handling instrument failures mid-read? Platform cross-validation (how is it conducted)? Carry-over (mitigation strategies)? A survey has been created to obtain community feedback on the different areas with open questions. Please go to the GBC website (team L3) to download the survey and send it to L3@globalbioanalysisconsortium.org. Let us know what group you are representing. A best practice document is currently being drafted and any major recommendations will be brought forward through GBC if appropriate. If you have any comments or opinions on the following section, these can also be sent to the L3 team.

6 Overview of suggested best practices Samples run in series versus on plates Calibration standards run first and QCs interspersed throughout the run Frequency of passing QC series determined during validation Suggested to be every samples Platforms affected: RIA, Biacore, Gyrolab, Singulex, & Immuno-PCR Carry over, hotspots and cross-talk effects Requires determination during method development Ideally, adjust conditions to eliminate effects. Platforms affected : Gyrolab (non-disposable tips), Biacore, Delfia, MSD, & Luminex Gyrolab or Biacore (carry-over): May require QCs to be placed more often if in series and another Calibration standard series after a certain number of samples Delfia (hotspots): run triplicate samples or use a white plate to run duplicates Luminex (cross-talk): change bead combinations to avoid cross-talk MSD (cross-talk): diminish the number of spots in the well or change antibodies Cell-based (cell contamination) : contamination of adjacent wells can be avoided through technique or placement of duplicates. 6

7 Overview of suggested best practices Multiplexing Validation criteria Each analyte is treated separately in terms of validation Effects of mixing different analytes is determined for recovery and precision Assay acceptance and data report: analyte specific Again, cross-talk must be avoided Sample analysis criteria When one analyte fails, run must be repeated. Partial sample run failure: accept samples within criteria Large dynamic range Increase number of calibration standards with a minimum of 3 per log of dynamic range May require more anchoring points for curve fit 2.5 QCs per log for validation and 1.5 QCs per log for sample analysis to keep the same ratio as with ELISA with the minimum number in accordance to the current guidelines Platforms concerned if typically give a 3-4 log dynamic range 7

8 Overview of suggested best practices for: Cell-based assays Special aspects of validation: 1. Controlling cell passages, plating and activity from experiment to experiment Visual or marker characteristics to validate use of cells Strict procedure for handling, plating and carrying out tests required once critical factors are defined F/T stability must be demonstrated; maximum number of cell aliquots prepared after one passage Effects of changing serum lots and other critical reagents must be shown. 2. Functional test requirements Control standard required to apply to all experiments to show expected functional effect High level of variability expected Treatment group can be run in parallel and normalized with control group Effects of individual sera may be high leading to difficulties of robustness 3. Further discussions ongoing... 8

9 Overview of suggested best practices for: FACS used for cell-based assay measurements Special aspects of validation: 1. Controlling preparation, staining and analysis from experiment to experiment Cells must be coming from a validated procedure for cell handling Fixing and staining of the cells must be done according to a validated protocol and tested for robustness to identify any variables leading to changes in staining intensity The necessary controls for controlling the antibody staining (primary and secondary antibodies), a non-stained control for gating purposes, an isotype control, and a positive staining control if possible to control between experiments. The FACs instrument must be validated and calibrated on a regular basis following an SOP Effects of changing serum lots and other critical reagents must be shown. 9

10 L3 GBC work group outcome The next section addresses the specific platforms in relation to acceptance criteria and pros and cons. The following platforms are covered: MSD Sector imager Biacore ie T200 RIA AlphaLisa Delfia Gyrolab Erenna (Singulex) Immuno-PCR Luminex Cell-based bioassays Any feedback on the slides are welcome and can be addressed to the L3 team at

11 Meso Scale Discovery (MSD) Micro titre plate based format utilising a electrochemiluminescence (ECL) signal. For PK measurements drug is bound using suitable antibodies in a format similar to ELISA. Electrochemical stimulation of Ru based detection molecules via plate based electrodes produces an ECL signal relative to the amount of bound drug. Drug

12 Key Differences in Assay Acceptance Criteria for the MSD Parameter Acceptance Criteria Hook Effect Reagent Stability Evaluation of a critical reagent Susceptable to Hook Effect, should be evaluated in dilutional linearity with relevent sample concetrations Reagents require specific labelling and are not commercially available therefore reagent stability should be evaluated New lots of labeled reagents should be assessed appropriately Plate Criteria Lot differences between plates can be assessed, especially when using streptavidin coated plates.

13 Meso Scale Discovery Advantages MSD reader has an increased dynamic range compared to classic spectrophotometer ELISA methods Potential for greater quantifiable range for analyte detection and reduced sample dilutions Disadvantages Single vendor system with relatively expensive reagents and single purpose instrument Potential Hook Effect, especially with 1 step methods Matrix interference and background signal is generally low leading to small MRD and increased assay sensitivity Assays can be performed using 1 wash step methods, which can increase sensitivity Criteria for validation of MSD methods can follow LBA regulatory guidance so no extra criteria are required Labeling of new antibody lots requires requalification Stability of labeled reagents should be investigated Can be used for any large molecule drugs where an antibody could be produced

14 MSD Multi-Spot Assay A MSD multi-plex assay format utilising MSD plates with multiple spots in each well. Each spot can be coated with a unique antibody to allow detection of multiple analytes simultaneously. 96 well 4 spot 96 well 10 spot 24 well 25 spot 24 well 100 spot

15 Key Differences in Assay Acceptance Criteria for the MSD multi-plex assay The MSD multi-plex assay includes all the differences noted on the general MSD assay slides plus the additional points below: Parameter Calibration Standards QC Samples Specificity/Cross Reactivity Acceptance Criteria PK assessments to follow ELISA criteria. Biomarkers included in the same analysis could have less stringent criteria. Potential for different curve fits and difficulty matching quantifiable range of all analytes at one MRD PK assessments to follow ELISA criteria. Preparation challenges for multiple analyte QC samples. Biomarker QCs included in the same analysis could have less stringent criteria Multiple analyte detection requires a wide range of critical assay reagents. Cross reactivity and specificity of the assay should be evaluated. Detection of drug product and binding target not possible in the same assay

16 MSD Multi-plex Assay The MSD multi-plex assay includes all the advantages and disadvantages noted on the general MSD assay slides plus the additional points below: Advantages Up to 100 spots available per well for different analyte detection Lower sample volume required to produce multiple data sets Disadvantages Potential cross reactivity of assay reagents. Drug and drug target can not be included together due to cross binding. Assay development challenges linked to detection requirements and sample dilutions required Potentially wide dynamic range of the MSD allows capture of the relevant concentrations of different analytes Commercially available kits for biomarkers and custom plate coating available for PK analytes from vendor Same equipment as used for MSD single analyte assays Challenging assay acceptance criteria and validation criteria due to multiple analytes Potential for large numbers of reagents so reagent stability considerations are important Plates coated by vendor so potentially not readily available and expensive If analyte detection requirements change does the whole assay need to be revalidated

17 Biacore For preclinical PK measurements, an anti-fc antibody can be immobilized on the chip surface for a generic assay that will bind all mab drug contained in the sample as it flows through the cell. The difference in light refraction due to drug binding, is read out in RU units. Other variations can also be used for specific assays such as target immobilized on the chip or an anti-idiotypic antibody. Presentation Title Presenter Name Date Subject Business Use Only 17

18 Key differences in assay criteria for Biacore Parameter Assay Validation Criteria Sample Analysis Criteria Carry over Cs and QCs Chip stability Robustness/ ruggedness Carry over should be measured over 10 cycles along and an expected maximum carry over effect that can be defined and incorporated into the signal change expectations. This can occur due to poor regeneration after each cycle. Run 3 times on 2 separate chips with QCs. If needed, a buffer blank can be subtracted in a reference flow cell. The expected range of signal decrease after each cycle based on the average decrease on all cycles during the validation and per chip, should be specified. Determine signal to noise ratio change after each cycle. Should be reasonable within i.e.6%. Need to also check absolute signal levels to follow the baseline. If a chip will be used for large studies: over 200 cycles, the long term reproducibility of the chip must be determined. QCs should be run 5 times each for a total of 100 cycles and a graphic to extrapolate expected signal decrease should be shown. From here, a minimum signal level cut-off can be determined. Signal change after each cycle should stay within a specified range. Add calibration standards at the beginning of the run and QCs throughout sample analysis. A new calibration standard curve should be added if the QCs fail. During sample analysis, signal decreases out of range should flag a warning for protein immobilization stability/ capacity to bind protein. Samples can be analyzed up to the point where signal decreases as defined in the validation.

19 Biacore Advantages Semi-automated (sample preparation manual) Generic assay can be run for comparing PK profiles of candidates Disadvantages Instrument is completely booked for one user at a time Expensive compared to regular immunoassay Can be GLP validated. Limited sensitivity Sensor chips are reusable Carry over can be a problem and must be evaluated to assure correct regeneration conditions. Reagents do not require labeling Multiplexing can be possible although development efforts required. Samples are measured immediately once they are passed over the chip Real time measurement.

20 RIA - Immunoassay Platform Radioimmunoassay (RIA) RIA (Radioimmunoassay) technique used for quantifying nanomolar and picomolar concentrations of hormones and other biomolecules in solution. 20

21 Assay Acceptance Criteria for RIA Parameter Acceptance Criteria Calibration Standards Larger dynamic range easy to optimize assay range for better curve fit. Run calibration standards for every parameter/ for every batch during validation Can interface with Watson LIMS. Quality Controls Run quality controls (ULOQ,HQC,MQC,LQC,LLOQ )in six independent runs during validation for precision and accuracy. Run quality controls (HQC,MQC and LQC) for every parameter/ for every batch during validation. Run quality controls (HQC,MQC and LQC) for sample analysis. Reagent Stability If custom kit is used stability of the kit reagents should be determined. Labeling of new batch might arise depending on half life of radio tag. Tube based runs Standards in duplicates can be run once and intermittent QC s in duplicates for every 50 samples can be maintained for a particular day run and consider as one analytical run.

22 RIA Advantages High sensitivity obtained using RIA Large dynamic range Can be GLP validated and interfaced with Watson LIMS Vendor does radio labeling both in custom kits and inhouse developed assays Disadvantages Constant manual operation involved for setting up an assay. Too many steps in Assay procedure steps involved Viz., precipitation, centrifugation steps Low throughput (maximum samples per day). Some sample pre-treatment may be required (filtration/centrifugation steps) Cost is high for kits and assay development

23 AlphaLISA Amplified Luminescent Proximity Homogeneous ImmunoAssay The platform utilizes a luminescent bead-based proximity format whereby singlet oxygen is channeled from an excited streptavidin-coated donor bead to an acceptor bead if in close enough proximity. The beads are brought into proximity through the use of a biotinylated capture antibody and a secondary detection antibody conjugated to the acceptor bead.

24 Key differences in assay criteria for AlphaLISA Hook effect Reagent stability Parameter Acceptance Criteria Susceptible to hook effect, should be evaluated in dilutional linearity 2 Ab conjugated Acceptor beads typically 1 year at 4 C (as per PE, depends on the shelf life of the molecule conjugated), cannot freeze 1x AlphaLISA Assay buffer only good for 1 week at 4 C SA-donor beads 1 year at 4 C, cannot freeze Critical and Non-critical reagents Plate criteria (ie border effects, lot differences) Critical: SA-Donor beads, Ab-Acceptor beads Non-critical: 10x AlphaLISA buffer, plates Have seen no edge effects or plate lot-to-lot difference Other Light sensitivity of donor beads matrix effect (quench of signal with high serum percentages) lot-to-lot differences in serum (seen in Biomarkers) 2 instruments to read (EnVision and Excite, both Wallac/PE)

25 AlphaLISA Platform Advantages Homogeneous, no-wash method Improved sensitivity over typical ELISA Disadvantages AlphaLISA Acceptor-bead conjugation costs higher than ELISA but similar to MSD (external conjugations, small-scale) Donor-bead light sensitivity Similar sensitivity to MSD methods Added challenge of manual pipetting into ½ area 96- well plates Short assay (2 hrs + sample prep time) Sole-source vendor Low sample volume (ca. 2mL) Can t eliminate matrix effect with wash High troughput since 384 well plates can be used Hook Effect is the strongest disadvantage

26 DELFIA Dissociation Enhanced Lanthanide Fluorescence ImmunoAssay The DELFIA platform utilizes the same sandwich assay fundamentals as a typical ELISA, but with a timeresolved fluorescence readout that takes advantage of a lanthanide chelate label (Europium) that has both a large Stokes shift and a high fluorescence intensity. Cartoon Adapted from

27 Key differences in assay criteria for DELFIA Hook effect Reagent stability Parameter Acceptance Criteria Susceptible to hook effect, should be evaluated in dilutional linearity. Can be avoided if run in a sequential format. No conjugations performed outside of biotin-ab, SA-Eu stability provided by vendor (6 months 1 year), cannot be frozen Critical and Non-critical reagents Critical: Streptavidin-Europium Non-critical: Enhancement Solution Plate criteria (ie border effects, lot differences) PE yellow plates prone to hotspots b/c of low background (autofluorescence), border effect not seen, but should be evaluated. Other 2 instruments to read (EnVision and Excite, both Wallac/PE) No issue w. SA plate or direct conjugation to Abs with SA

28 DELFIA Platform Advantages Comparable dynamic range Low matrix effect (low MRD) Disadvantages Throughput like ELISA Possible auto-fluorescence of plates Better sensitivity by comparison to our ELISA Sole-source vendor Theoretical possibility of multiplexing with different lanthanide chelate labels High signal to noise level

29 Gyrolab xp workstation automated immunoassay in CD Fluorophorelabeled detection reagent Reagent channel Sample chamber, 20, 200 or 1000 nl Analyte Biotin-labeled capture reagent Streptavidin coated beads All assay steps automated inside instrument Samples, standards, QCs, reagents loaded in 96-well MP Samples and reagents transferred to CD with liquid transfer device Sample and reagent nanoliter volumes defined by CD spinning Laser-induced Fluorescens detection Automated sample processing in Gyrolab xp workstation Possible to run up to 5 CDs in unattended run 1 CD 1 hours, 5 CDs 5 hours 15 nl affinity bead column One microstructure: one data point 8 microstructures/ segment segments/ CD

30 Key Differences in Assay Acceptance Criteria for the Gyrolab xp workstation Parameter Calibration Standards Quality Controls Multi-CD- runs Carry over Other Acceptance Criteria Larger dynamic range more standard points could improve curve fit Run calibration standards in six independent runs during validation. 1 run = 1 CD Option to include standard curve once per multi-cd-run if sample/reagent stability in multi-cd-runs shown during development/validation Run quality controls in six independent runs during validation. 1 run = 1 CD For multi-cd-runs, determine stability of samples/reagents, by including calibration controls and QCs on each CD. Use volume in MP planned to use during sample analysis Test early during development to confirm no quantifiable levels of carry over for the assay and intended range. Plate criteria CD is circular, across CD precision only looked at if a specific need arises One batch of labelled reagents tested during validation. New batch cross validate

31 Gyrolab xp workstation Advantages Fast assay run time (1 hour + sample prep) and assay development time Less matrix interference in serum/plasma, Small sample and reagent volume required Automated walk-away capability, the immunoassay portion is hands-off for technichans Can be GLP validated and interfaced with Watson. Software and is 21CFR part 11 compliant. Regulatory support available. Can run up to 5CDs in a row and possibly envision a run over multiple CDs. Easy to run multiple assays at the same time Automation improves throughput and enhances data quality Broad dynamic range and increased sensitivity of immunoassay Disadvantages Sample stability should be evaluated to confirm stability in multi-cd-runs Capture and detection reagents need to be labeled, (many generic reagents are available commercially pre-labeled) Carry-over needs to be evaluated/optimized Only one vendor Buffer components are not disclosed and it is unknown what can interfere with analytes that are sensitive to certain buffers Not possible to multiplex (nl volume requirements allow multi-sampling) Cost of consumables

32 Singulex Erenna Immunoassay Platform 2-step process to achieve maximum sensitivity and minimum non-specific binding: Step 1: Execute a microparticle (MP)-based sandwich immunoassay Step 2: Elute the sample and utilize a unique single molecule counting technology Dye-labeled anti-drug antibody (Detector) MP coated with antidrug capture Ab Elute dye-labeled detector/ transfer to 384-well plate Plasma sample MP Count single molecules with Erenna System MP Incubate and wash MP STEP 1: Sandwich Immunoassay 32 STEP 2: Single Molecule Counting

33 Key Differences in Assay Acceptance Criteria for Singulex Erenna Parameter Calibration Standards Quality Controls Reagent Stability Multi-plate runs Acceptance Criteria Larger dynamic range more standard points needed Vendor software has unique curve fitting algorithm using three different readout types Vendor recommends clarification of samples and QC s using filtration or centrifugation -Test effect of pretreatment in validation could get loss of recovery Assay reagents are modified by the vendor and assays supplied as a custom kit, therefore stability of the entire kit should be determined: -Most kit components are stored at 4 o C (capture reagent coupled microparticles cannot be frozen) Option to run standards on first and last plate and QC s on every plate (test during validation)

34 Singulex Erenna Advantages Ultra-sensitivity may be obtained using this format (very low pg/ml fg/ml levels are achievable) Large dynamic range Can be GLP validated and interfaced with Watson LIMS Vendor does all critical reagent labeling and assay development and supplies custom kits Disadvantages System is not automated, no walk-away capability (some assay steps may be automated with a liquid handler) Assay procedure involves many wash/transfer steps Low throughput (4 x 96-well plates maximum per day) Some sample pre-treatment may be required (filtration/centrifugation steps) Cost is high for kits and assay development

35 Immuno-PCR Platform

36 Key Differences in Assay Acceptance Criteria for the Immuno-PCR Parameter Calibration Standards Acceptance Criteria Broad Dynamic range, may need more claibration standards than normal ELISA to cover the range Reagent Stability Evaluation of a critical reagent Reagents require specific DNA- conjugation and are not commercially available through multiple vendors therefore reagent stability should be critically evaluated New lots of labeled reagents should be assessed appropriately Plate Criteria Lot differences between plates should be assessed, currently the top-yield modules are also not available commercially through multiple vendors

37 Immuno-PCR Advantages Ultra-sensitivity may be obtained using this format (very low pg/ml fg/ml levels are achievable) Potential for greater quantifiable range for analyte detection and reduced sample dilutions Disadvantages Single vendor system with relatively expensive reagents, limited CRO s to test clinical samples Minimization of reagent/instrument/workplace cross contamination as any other PCR method using filter tips and other precautions Matrix interference and background signal is generally low Higher MRD while keeping the assay sensitivity due to PCR amplification of signal Labeling of new antibody lots requires requalification Stability of conjugated reagents should be investigated Assay longer due to PCR step 1-2 hour after ELISA Data analysis cumbersome as compared to ELISA Data analysis bit cumbersome as compared to ELISA

38 Luminex 38

39 Key differences in assay criteria for Luminex Parameter Acceptance Criteria Calibration standards QC s Curve fitting parameters Parallelism ISR In-plate standard preferred Multiple standards for different analytes Anchoring points vary for different analytes Practice is inconsistent among analytes Multiple QC s QC s are typically made by analytes in plasma with recombinant proteins or to use plasma that contains endogenous targets; QC s are often stored frozen. Curve fitting option is dependent on vendors. 4P and 5P with weighing options are generally available. Because of multiplexing nature, curve fitting will also depend on individual analyte Typically assessed by looking at dilution linearity using samples that contain endogenous analyte and compare it with recombinant proteins. This is due to the primary application of the technology in PD/biomarkers rather than PK. Analyte-dependent, and therefore challenging to set acceptance criteria. Practice is inconsistent among analysts. Some will mask data if needed

40 Luminex Advantages Multiplexing capability, Low cost (for sampling multiple analytes) Low sample Volume (for sampling multiple analytes) Commercially available kits for some analytes Instrument validated 21CFR part11-- BioRad Disadvantages Low throughput (especially if the bead is polystyrene-based and a filter plate is needed) High maintenance requirement for instrument Cross-talk potential Assay acceptance criteria challenging to establish due to multiple analytes Reagents are light-sensitive

41 Cell Based Bioassays 41

42 Key differences in assay criteria for Cell Based Bioassays Parameter Acceptance Criteria Replicates QCs Specificity ISR Cs and QCs Duplicates as default. Triplicates may be needed based on assay performance. LLOQ, LQC, MQC, HQC, and ULOQ precision and accurracy criteria up to 30% and total error up to 40% may be required. Additional cell line performance parameters such as signal, confluency, and viability should be considered as appropriate. Cellular target may be activated by multiple endogenous ligands. Selectivity assessment in >10 individual matrices from healthy and target population recommended. %difference of the original to reanalyzed result up to 40% may be required. Under current guidances it is not clear if ISR is required for cell based PK assays. Add calibration standards at the beginning of the run and QCs throughout sample analysis.

43 Key differences in assay criteria for Cell Based Bioassays (continued) Cell Stability Other Parameter Plate criteria (ie border effects, lot differences) Acceptance Criteria Cell passage and freezing stability need to be assessed. Within the same run, QC performance (%RE 30%) assessed against calibration standards in both fresh and stability cells. Ranges for parameters such as assay signal, cell growth rate, and viability should also be considered as appropriate. Batched working banks performed under single campaign may require qualification of each batch. New working banks established outside of validation require qualification. Critical Reagents: assay performance with multiple FBS/FCS lots/sources should be evaluated. Media and supplement sources may also impact assay performance. Plate based assays susceptible to edge effects and requires evaluation

44 Cell Based Bioassays Advantages Measures bioactive drug Alternative source for target protein if unavailable Disadvantages Resources Specialized staff, facilities, storage Increased development, validation, analysis costs Cell line Identification and generation Freedom to operate concerns : intellectual property and licensing Cell line performance May be poor with human matrices Edge effects on plates Propagation and maintenance Poor robustness/ruggedness and challenging to transfer

45 Bioactivity Assays (i.e. Coagulation, Enzymatic) COAGULATION PATHWAY 45

46 Key differences in assay criteria for Bioactivity Assays Parameter Calibration Standards QCs Stability ISR Other Acceptance Criteria The simplest model that adequately describes the concentrationresponse relationship. Historical practice of using linear regression models. LLOQ, LQC, MQC, HQC, and ULOQ precision and accurracy criteria up to 30% and total error up to 40% may be required. Thermal and F/T instability expected. Based on F/T instability, sample volume limitations, and assay performance, ISR extremely challenging. %difference of the original to reanalyzed result up to 40% may be required. Critical reagents: aptt and PT sources, instrumentation, immunoabsorbed vs congenitally deficient plasma Different plasma lots may significantly impact assay performance. New lots require qualification and may require partial validation. Inhibitors may be present in some patients. Coagulometer may require procedural controls to meet 21CFR11 compliance

47 Bioactivity Assays (i.e. Coagulation, Enzymatic) Advantages Measures bioactive drug Disadvantages Really a PD assay surrogate for PK Automated instruments Complexity of coagulation biopharmaceuticals and assays High inter-patient variability due to contribution by other clotting factors and inhibitors Thermal and F/T instability Discrepancies between source of International Standards vs. current recombinant products Discrepancies between FDA and EMA requirements for product release test methods and potency assignments Limited external support (human PK analyses at fully accredited coagulation labs are no longer acceptable High assay variability observed between laboratories (20-40% CV)