Practical Aspects of Dried Blood Spot (DBS) Bioanalytical Assay Development and Application. Qin C. Ji CASSS, 09 September 2010

Transcription

1 Practical Aspects of Dried Blood Spot (DBS) Bioanalytical Assay Development and Application Qin C. Ji CASSS, 09 September

2 Outline Introduction Potential advantages of DBS DBS assay development Result of an example DBS TK study DBS assay validations 2

3 What is Dried Blood Spot (DBS) Technology? It was introduced in Scotland by Robert Guthrie in 1963 and is widely used for newborn screening and other disease testing. Blood samples are collected on DBS cards, then dried and stored for analysis. During sample analysis process, a disk of specific diameter will be punched out. DBS cards are made from either chemically treated or untreated filter papers. DBS card can be made in different physical format. 3

4 Characteristics Which Make DBS a Good Alternative to Current Sampling Process Small sample volume usable with sensitive detection technology No need for accurate measurement of the blood volume Amount of drug in a consistently punched spot size should be the same for blood samples with same drug concentration. Some compounds exhibit much improved storage stability at room temperature 4

5 Benefits in Drug Development Ethical and social benefits Financial benefits Reduced costs for storage and transportation For tox studies Animal numbers Procedures Test substance Scientific benefits Better data quality for tox studies Serial vs. composite TK from central study animals 5

6 Benefits in Drug Development Operational benefits Enable studies at the remote locations where centrifuges, freezer or refrigerators are not available Improve patient recruitment for clinical studies Bioanalytical Benefits No need of uncap and re-cap sample vials Potentially fewer assay issues related to drug adsorption to the sample vials and solubility or precipitation of the compounds 6

7 Effect of Different Volumes of Blood Applied to the DBS Cards Dextromethorphan Dextrorphan Conc. (ng/ml) microliter blood sample Accuracy (%) Precision (%) microliter blood sample Accuracy (%) Precision (%) microliter blood sample Accuracy (%) Precision (%) N= 6 for all QCs Liang, X., Li, Y., Barfield, M., Ji, Q.C., J. of Chromatogr. 877,

8 Assay Precision and Accuracy of Using Center Spots and Non-center Spots Dextromethorphan Dextrorphan Conc (ng/ml) Center Accuracy (%) Precision (%) Non-center Accuracy (%) Precision (%) N= 6 for all QCs Liang, X., Li, Y., Barfield, M., Ji, Q.C., J. of Chromatogr. 877,

9 Measured Conc. vs. Blood Spot Area Measured concentration (ng/ml) DM DT I I II II I : 40 ng/ml II : 10 ng/ml DBS spot square diameter (mm 2 ) (Measured concentrations based on a standard curve with 3 mm punch size) Liang, X., Li, Y., Barfield, M., Ji, Q.C., J. of Chromatogr. 877,

10 Stability on Card at Room Temperature (DM and DT) 107 days Fresh Stored Dextromethorphan (DM) Concentration (ng/ml) Accuracy (%) Precision (%) Dextrorphan (DT) Accuracy (%) Precision (%) N= 6 for all QCs Liang, X., Li, Y., Barfield, M., Ji, Q.C., J. of Chromatogr. 877,

11 On-line Sampling (Direct Analysis) Multiple emerging technologies for direct analysis of DBS samples CAMAG TLC-MS Interface Desorption Electrospray Ionization (DESI) Mass Spectrometry Liquid Extraction Surface Analysis (LESA) System Advantage of on-line sampling (direct analysis) No need of card punching Efficient utilization of samples Potentially high throughput Not available for practical use yet 11

12 Off-Line Sampling (DBS Card Punching) Currently, it is a more practical approach. Punching disks out for further sample analysis has been implemented in clinical testing labs for many years. After sample spots are punched out from DBS cards, sample preparation is a parallel process. Potentially higher throughput Current knowledge about sample preparation is a good basis to start. Further investigation of the extraction is necessary due to different sample format. Potentially cleaner sample extracts 12

13 Automation for DBS Assays BMS evaluations of the BSD600 Has been used for clinical labs for many years, good mechanical design 96 samples punched out in about 15 minutes Barcode reading to record sample location in a 96-well plate Further improvement needed IQ/OQ requirement for regulated studies Audit trail for compliance Interference with LIMS system for sample sequence loading Further improvement of user interface Exhaust management for potential dust generation 13

14 DBS Extraction Methods Organic Extraction (OE) 200 µl Dry and IS (in 50 µl ACN or MEOH) µl ACN or MEOH Reconstitute DBS (punched 4-mm disk) Protein Precipitation (PPT) ACN (500 µl) IS (in 100 µl H 2 O) 400 µl Dry and Reconstitute Liquid-Liquid Extraction (LLE) 400 µl Extraction Solvent (600 µl) IS (in 50 µl H 2 O) µl buffer Dry and Reconstitute Liu, G., et. al., Bioanalysis 2(8), ,

15 Matrix Ion Suppression/Enhancement after LLE and PPT 1.3e5 FTA elute Neat solution LLE PPT Intensity, cps 3.0e5 FTA classic Ion suppression Ion Enhancement 2.4e5 903 Card Time, min Liu, G., et. al., Bioanalysis 2(8), ,

16 Elution Efficiency Elution efficiency (%) PPT ACN 903_L PPT ACN 903_H PPT MeOH 903_L PPT MeOH 903_H OE ACN 903_L OE ACN 903_H OE MeOH 903_L OE MeOH 903_H LLE 903_L LLE 903_H LLE FTAc_L LLE FTAc_H Omeprazole PPT ACN 903_L = Protein precipitation by acetonitrile using a 903 Protein Saver DBS card spotted with low concentration (100 ng/ml) blood sample LLE FTAc_H = Liquid-liquid extraction using an FTA Classic DBS card spotted with high concentration (1000 ng/ml) blood sample Liu, G., et. al., Bioanalysis 2(8), ,

17 Omeprazole DBS Assay Sample collection Spot 15 µl L whole blood onto Whatman 903 card. Allow to dry on benchtop for ~2 hr. Store in a ziploc bag with desiccant packets at room temperature. Sample Procession Punch out 4-mm 4 disk from standard, QC and sample into 96-well plate. Add IS and buffer solution, then sonicate for 5 min. Add acetonitrile for protein precipitation Supernatant used for LC-MS/MS analysis Sample Name: "16037 Newtechnique evaluation LLOQQC0.14 1" Sample ID: "37" File: "snapph_ _16_dbs-omp_a&p2_.wif" PeakName: "Omp" Mass(es): " / Da" Comment: "none" Annotation: " Sample Index: 37 Sample Type: QC Concentration: ng/ml Calculated Conc: ng/ml 1500 Acq. Date: 4/28/2010 Acq. Time: 7:00:34 PM Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 8 Noise Threshold: 4.34 cps Area Threshold: cps,num. Smooths: 5 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: sec Expected RT: min Use Relative RT: No Int. Type: Manual Retention Time: min Area: counts Height: cps Start Time: min End Time: min I n t e n s i t y, c p s Time, min Sample Name: "16037 Newtechnique evaluation LLOQQC0.14 1" Sample ID: "37" File: "snapph_ _16_dbs-omp_a&p2_.wif" PeakName: "IS-Omp(IS)" Mass(es): " / Da" Comment: "none" Annotation: " Sample Index: 37 Sample Type: QC 0.55 Concentration: ng/ml 1.3e5 Calculated Conc: N/A Acq. Date: 4/28/2010 Acq. Time: 7:00:34 PM 1.2e5 Modified: Yes Proc. Algorithm: Analyst Classic Bunching Factor: 7 Noise Threshold: 8.05 cps Area Threshold: cps,num. Smooths: 5 Sep. Width: 0.20 Sep. Height: 0.01 Exp. Peak Ratio: 5.00 Exp. Adj. Ratio: 4.00 Exp. Val. Ratio: 3.00 RT Window: sec Expected RT: min Use Relative RT: No I n t e n s i t y, c p s Int. Type: Base To Base Retention Time: min Area: counts Height: cps Start Time: min End Time: min uhplc-ms method LEAP 4x Ultra uhplc with Run time of 1.5 min AB Sciex API 5500 with Electrospray Positive ionization mode Snapp, H., et. al., ASMS e5 1.0e5 9.0e4 8.0e4 7.0e4 6.0e4 5.0e4 4.0e4 3.0e4 2.0e4 1.0e4 LLOQ 0.1 ng/ml RT 0.56 min d3-omeprazole 0.5 ng/ml RT 0.55 min Time, min 17

18 Comparison of TK Curves Obtained from Three Measurements (Omeprazole DBS TK Study) Concentrations (ng/ml) with SEM DBS Plasma Blood Time (hr) Plasma data corrected for blood:plasma ratio Snapp, H., et. al., ASMS

19 Validations Sample collection Quality control of the DBS card prior sample collection Temperature, Humidity Environment during sample collection Temperature, Humidity Variation of blood volume deposited on the card Variation due to different batches of card DBS sample drying conditions and duration Standard and QC preparation Drug distribution prior to spotting blood standards and QCs onto the DBS cards Variation of blood samples Hemolysis Temperature of the blood when deposited onto the card 19

20 Validations Storage stability Temperature range Durations of sample storage at different temperature Carry over Card punching carry over Detection system carry over Dilution Matrix effect Recovery Selectivity Accuracy and precision 20

21 Validation (Fits for Purpose) Pre-clinical studies More control, less variables for validation Fixed blood volume More defined temperature range Use same lot of sample collection card Clinical studies More flexible, more variables for validation Cover most of variation discussed in previous slides Don t t expect to work for all compounds and all studies Some of validation requirements could be removed as the industry has more experience with this technology. 21

22 Summary DBS is an emerging technology in the field of pharmaceutical development that could significantly impact future bioanalytical processes. Rapid progress has been made in understanding this technology for practical usage. High throughput DBS bioanalysis will be achieved with the further improvement in automation and/or in utilization of direct injection. Validation and qualification of the assay need to consider all of the related scientific aspects of this technology. A Fit for purpose strategy could be used to balance the investment in assay development and sample collection. 22

23 Acknowledgments Guowen Liu Heidi Snapp Celia D'Arienzo Laura Patrone Alicja Batog Jack Valentine Mark E. Arnold Adrienne Tymiak Tim Olah Michael Graziano BMS DBS Discussion Group 23