easypunch STARlet: Increasing throughput and lowering sample volume in DMPK studies by automating Whatman FTA TM DMPK DBS card processing

Transcription

1 easypunch STARlet: Increasing throughput and lowering sample volume in DMPK studies by automating Whatman FTA TM DMPK DBS card processing Application note Top 3 reasons for automation of this assay FTA DMPK cards are a major improvement for blood sample analysis in drug metabolism and pharmacokinetic studies. They greatly reduce the blood volume required for tests and make storage and shipment of samples very easy. To exploit their potential to the full, automated procedures for DMPK card processing are required. Ensure easy and safe processing of dried blood samples. Reliably punch and extract samples from FTA DMPK DBS cards. Provide full traceability of sample during processing. Introduction Drug metabolism (DM), pharmacokinetic (PK) and toxicokinetic (TK) studies provide crucial insight into how drug candidates behave in the body and are therefore critical steps in drug development. These types of analysis are time-consuming and limited in throughput. Traditionally, they require large volumes of blood to provide sufficient volume for quantitative bioanalysis. Another limitation is the sampling method. Since only a limited number of serial samples can be taken from each experimental animal, composite sampling is often used, resulting in lower quality PK data and an increase in the number of animals required. Finally, there are practical challenges with shipping and storing blood samples, which require controlled handling, frozen transportation and storage. The dried blood spot (DBS) method is an alternative technique that overcomes these drawbacks. Widely used to screen for metabolic problems in newborn babies, it is now being successfully employed in PK studies. FTA DMPK cards are used to collect dried blood spots. One of the bottlenecks identified in switching from plasma to DBS analysis has been the lack of automation available to both punch and extract DBS samples. In collaboration, GE Healthcare and Hamilton Robotics have developed an automated system called easypunch STARlet (see Fig. 1) that is capable of processing DBS samples, from recognizing dried blood spots on FTA DMPK cards to punching samples and extracting them for analysis. Figure 1: Hamilton s easypunch STARlet. Partner Life Sciences

2 FTA TM sample collection card punching and DBS sample extraction - all in one Hamilton s easypunch STARlet is an integrated system for punching and pipetting which allows for reliable DBS card processing, from detecting and analyzing the dried blood spot to preparation of samples ready for LC/MS analysis. Method description FTA DMPK cards are imaged and the best position for punching in relation to the sample spot is determined. A sample is punched into a well of a receiving plate and the position of the punched DBS disc in the plate is checked. Liquid is added to the individual wells for the extraction of the DBS punch. A detailed description of the method is shown in the DBS punching protocol. Preparation Punching Extraction DBS punching protocol Load FTA TM DMPK cards into the magazine racks on the deck layout. Load receiving plates onto the punching unit. Transfer FTA TM DMPK card from its position in the magazine to the imaging position on the punching station. Take an image of the card. Read barcode and analyze the sampling area. Determine the best position for punching in relation to the sample spot. Transfer FTA DMPK card to the punching position. Punch a sample into a well of a receiving plate. Take a post-punch image of the receiving plate. Check position of the punched DBS disc in the plate. Transport the receiving plate back to its original deck position. Add liquid to the individual wells for extraction of the DBS punch. Technology Figure 2: Visual workflow. FTA TM DMPK cards for collecting blood FTA DMPK cards (Fig. 5) use specialized Whatman media for sample application directly onto a paper matrix (see Barfield et al.). This allows fast and easy collection of bio-fluids such as whole blood, plasma or urine. The FTA DMPK-A and FTA DMPK-B cards lyse cells and denature proteins on contact while FTA DMPK-C cards do not carry any chemicals and leave the samples in non-denatured state. Indicating versions of the cards allow for easy identification of clear biosamples, such as plasma. Punching and imaging unit Hamilton s easypunch system includes a module with punching and imaging functionality (Fig. 3). It consists of a charged couple device (CCD) digital camera, and a punch unit with interchangeable punch head. The camera is mounted on top of the punching unit, together with the illumination source. It is used for imaging of cards for determination of the punch position and of plates which receive the punches for process control. Punch heads with different punch diameter sizes, e.g. 1.2, 2, 3 and 6mm, are available. A release button allows easy removal for the exchange or for cleaning of the punch head. Sample tracking Figure 3: Punch unit with punch head and light table. The Autoload function of the Microlab STARlet enables automatic loading of all labware and reading of the barcodes on both the plates and magazines. The barcode on each card is read during the punching process. It is connected to the position in the magazine and the position of the punch in the receiving plate which is verified by imaging. The output file containing this information can be imported into a Laboratory Information Management System (LIMS). This allows full traceability of all samples within the system. Cards may be stored in the magazines until the result is verified. If an error has occurred with individual cards, information on the position of the card could be used to reprocess these cards.

3 Protocol System description The easypunch platform is a Hamilton Microlab STARlet system comprising of four pipetting channels on a MPH/iSWAP robotic arm. Two modules are included, one that transports and the other that punches FTA DMPK cards. The deck contains magazines for holding FTA DMPK cards, receiving plates for punching into and positions for reagents and tips. The magazines have been designed to hold FTA DMPK cards presented to a specific gripper. This card and plate gripper (Fig. 4) is located at the MPH extension of the MPH/iSWAP pipetting arm. It is used to transport cards and plates to the punching station and back. The punching unit is located on the left hand side of the system covering tracks 1-6. A plate slide features two plate positions for Deep Well or Microtiter Plates which receive the DBS punches. Liquid is added to the individual wells for the extraction of the DBS punch using the pipetting channels of the system. A waste collection point between the plate positions can be used for disposal of cleaning punches which are used to minimize carryover. Alternatively, one of the plate positions can be used for waste. An integrated vacuum system removes dust. A deionizer can eliminate problems resulting from static charge of punches. A plastic cover is used for end user safety. Figure 5a: Indicating FTA TM DMPK cards. When a sample is applied the blue dye is displaced, leaving a lighter-colored area for easy identification. Figure 4: Card and plate gripper. Application software Figure 5b: Non-Indicating FTA TM DMPK cards. Hamilton s easypunch software allows for superior card imaging and analysis with full traceability of samples. It enables recognition of positively stained samples (e.g. blood on a non-indicating card) and negatively stained samples (e.g. plasma on indicating cards). Cards with sample are defined by over a 100 parameters, including roundness and minimal spot size. An algorithm analyzes defined areas of the card image for different features such as card layout, position of the gripper relative to the card and barcode. In the sampling area, the software will identify samples and determine the best punching position according to preset parameters (Fig. 6). Barcode recognition is used for sample identification. Already punched positions on the card are recognized. After punching of a card, an image of the receiving plate is taken to analyze the position of the punched discs in the wells. Recognized wells are labeled by green circles, detected punches are highlighted by white spots, and wells with no punch detected are labeled by a red cross. Figure 6: Image analysis and determination of punch position. Green area: determined sample position; blue circle: previously punched position; red circle: determined punching position.

4 Application testing using the easypunch STARlet Dried blood spots were prepared by spiking Human blood with various concentrations of Simvastatin, a hypolipidemic drug. Physicochemical properties of Simvastatin are shown in Table 1. The anticoagulant used was citrate phosphate dextrose. Stock solutions of Simvastatin and its D6 deuterated internal standard were prepared in 90:10 (v/v) methanol:deionised water. Working solutions were prepared in 25:75 (v/v) methanol:deionised water. The stock solutions were diluted 1 in 10 by spiking them into blood. The mixture was pipetted up and down 5 times to allow mixing. Calibration standards were prepared at 0.5, 1, 2, 4, 8 and 10µg/ml. Replicates (n=5) were prepared at 0.5, 1.5, 5 and 7µg/ml. Aliquots of Human blood (20µl) were spotted onto FTA DMPK-A chemistry based cards. Cards were placed in racks enabling them to stand upright and dried thoroughly for 2 hours. The easypunch STARlet system was programmed to punch one 3mm diameter punch from the centre of each sample into the individual wells of a 96 well polypropylene plate. One cleaning punch was taken from a blank card after each blood-containing punch. After all punches were taken, the liquid handling functionality of the easypunch STARlet system added 100µl of extraction buffer; 90:10 (v/v) methanol:deionised water containing 0.5µg/ml of internal standard to each well. The plates were sealed with a cover plate and were transferred to an orbital shaker set to 150rpm, where it was agitated for 1 hour. 2µl of supernatant from each well was then injected onto a LC-MS system. A total of three punching and extraction runs were carried out using the easypunch system. The percentage coefficient of variation (% CV) was calculated based on the analysis of n=5 replicates at four concentration levels (0.5, 1.5, 5 and 7µg/ml) per run. Both inter and intra batch % imprecision were calculated. The analyte concentration in the blank card (positioned directly after the highest concentration calibration standard) was also evaluated. Compound structure Chemistry Mwt LogD (ph7.4)=4.7 Neutral Table 1: Physiochemical properties of Simvastatin

5 Results Imprecision testing (Table 2) revealed that the intra and inter % CV was within ±20% when Simvastatin was spiked into human blood at the LLOQ (lower limit of quantification), low, middle and high concentration levels. Chromatograms of human blood extracts spiked at 0.5µg/ml and 10µg/ml and of a blank containing internal standard monitoring Simvastatin and deuterated internal standard are shown in Fig. 7. No significant levels of carryover were observed based upon the response of Simvastatin in the blank after injection of the highest calibration standard relative to the response of Simvastatin in the lowest calibration standard. Simvastatin concentration (µg/ml) LLOQ LOW MIDDLE HIGH 0.5µg/ml 1.5µg/ml 5µg/ml 7µg/ml Batch 1 % CV Batch 2 % CV Batch 3 % CV % CV intra-run , % CV inter-run Table 2: Results: % CV testing Figure 7a: blue peak = 10µg/ml Simvastatin red peak = D6 deuterated Simvastatin internal standard Figure 7b: Blank spiked with internal standard Figure 7c: 0.5µg/ml Simvastatin with internal standard

6 Conclusion The easypunch STARlet system facilitates both the automated punching and extraction of dried blood spots using FTA DMPK cards. The low intra and inter batch variation of the test samples demonstrated that this powerful system is suitable for high throughput DMPK studies. It is capable of combining microvolume sampling with robust and reliable automated sample processing. The system offers full flexibility to add plate shakers, centrifuges etc., thus allowing the complete automation of a typical DMPK extraction workflow. Reference: Barfield M et al.. Use of dried plasma spots in the determination of pharmacokinetics in clinical studies: validation of a quantitative bioanalytical method. Anal Chem Jan 1;83(1): System requirements Part number easypunch STARlet System dimensions: Width: 1124mm Height: 903mm Depth: 1006mm Labware requirements Part number / Provider FTA DMPK STARTER PACK FTA DMPK-A IND Cards (indicating, blood spot area is ~20% smaller than on DMPK-B or DMPK-C cards, denaturing, impregnated) FTA DMPK-B IND Cards (indicating, denaturing, impregnated) FTA DMPK-C IND Cards (indicating, non denaturing, no impregnating chemicals added) FTA DMPK IND STARTER PACK FTA DMPK-A Card (blood spot area is ~20% smaller than on DMPK-B or DMPK-C cards, denaturing, impregnated) WB / GE Healthcare WB / GE Healthcare WB / GE Healthcare WB / GE Healthcare WB / GE Healthcare WB / GE Healthcare FTA DMPK-B Card (denaturing, impregnated) WB / GE Healthcare FTA DMPK-C Card (non denaturing, no impregnating chemicals added) WB / GE Healthcare High vol. CO-RE tips, µl 96-well polypropylene plates / Hamilton 3364 / Costar Partner Life Sciences Web: USA: infoservice@hamiltonrobotics.com United States Tel: United Kingdom & Ireland Tel: +44 (0) Brazil Tel: +55 (11) Hamilton Robotics GmbH. All rights reserved. All trademarks are owned and/or registered by Hamilton Bonaduz AG in Switzerland and/or other countries. Lit. No. AN /01 QTY: 600, 08/12 Printed in Germany. China Tel: France Tel: +33 (01) Italy Tel: To find a subsidiary or distributor in your area, please visit hamiltonrobotics.com/contacts. Denmark, Norway, Sweden, Finland Tel: +46 (0) Germany, Switzerland, Austria, Benelux Tel: +49 (0)