The exploding number of potential drug targets emerging from molecular biology research has led to the need to create large and diverse compound libraries and test them for pharmaceutical activity. To meet demand for new leads, the generation of synthetic chemical libraries is required to replace the dwindling and difficult to purify natural sources used in the past. Chemical diversity can be created using combinatorial methods and screened using high throughput methods, thereby identifying the most promising "lead" pharmaceutical compounds. To assist in the discovery of pharmaco-active structures, two technology platforms have emerged which enhance the creation and purification of complex compound libraries using informatics-driven combinatorial methods. First, the generation of new materials and devices for the chemical synthesis and purification of compound libraries; and second, robotic liquid handling to facilitate massively parallel processing. 

Materials and devices-Due to the use of harsh chemicals in many of the processing steps, there is a need to develop devices that not only resist chemical degradation but also do not shed unwanted extractables. New devices are needed for the synthesis and purification of compounds under a variety of chemical environments.

Automated processing-Robotic handling is required for processing the large libraries in synthesis, cleavage and harvesting steps. This has created additional requirements such as robotic compatibility for hands-free operations, crosstalk elimination, bar coding for informatics, low weeping for contamination control and consistency for reliable purification.

• Low binding and chemically resistant polypropylene membrane
• Individually sealed membrane disks in each well
• Polypropylene plate assembly
• Standardized well spacing for automated fluid handling
• Single piece construction for rigidity and robotic handling
• Unique serialized bar code

Low Extractables

Filtration media used for combinatorial chemistry synthesis or cleavage applications is designed to retain the solid-phase beads while allowing the solution phase to pass during the wash steps. The incorrect selection of filtration media or housing material can be a significant source of extractable contaminants which can interfere with downstream binding assays, chemical cleavage or subsequent chemical analysis. Pall Life Sciences GHP (hydrophilic polypropylene) membrane was developed with a patented grafting process which does not shed. This membrane has been used widely for analytical chemistry filtration applications where chemical resistance and high purity materials are required to ensure low extractables. Both the GHP membrane and the polypropylene housing of AcroPrep 96 filter plate carry HPLC certification for low levels of UV-absorbing extractables.  

Chemical Compatibility

Chemical resistance is a major consideration when selecting the right filter plate housing and filter media for combinatorial applications. Pall Life Sciences GHP separation media offers excellent chemical compatibility for cleavage applications and has been used for a variety of aqueous, acidic, basic, non-aggressive organic and aggressive organic solutions (Table 1).

Chemical compatibility is compromised when the fluid adversely affects the integrity or efficacy of the filtration media or housing resulting in high variability, melted housings, leakage, compromised filtration or extractable contamination of the sample. A high grade of polypropylene was selected for the housing of AcroPrep 96 filter plate to ensure that the material would offer a high level of chemical compatibility for a wide range of solutions while maintaining integrity of analytical results.

 

The data presented in this chart is a compilation of testing by Pall Life Sciences with certain chemicals, manufacturer's data, or compatibility recommendations from the Compass Corrosion Guide, by Kenneth M. Pruett. This data is intended to provide expected results when filtration devices are exposed to chemicals under static conditions for 48 hours at 25 °C (77 °F), unless otherwise noted.

Automation Compatibility

The AcroPrep 96 filter plate was designed with automation in mind. As such, it follows Society for Biomolecular Screening (SBS) guidelines for plate dimensions, well volume and height, and well-to-well spacing. This allows the AcroPrep 96 filter plate to be compatible with a variety of liquid handlers currently on the market. In addition to these automation features, the AcroPrep 96 plate is a rigid single piece unit, which enables it to be used with a variety of robotic arms. Finally, each AcroPrep 96 plate comes with a unique bar code on the side, thereby allowing computerized tracking.  Thoughtful design ensures that AcroPrep 96 filter plates can be used successfully with automation equipment from the following manufacturers: Adept Technology Inc., Beckman, Cyber Lab, Hamilton Company , Agilent Technologies, Matrix Technologies Corp., Oyster Bay Pump Works, Inc., PerkinElmer Inc., Porvair, Qiagen Inc., Robbins Scientific Corp., Robocon, Tecan U.S., Titantele, Tomtec Inc., Velocity 11, and Waters, Corporation.

Solution Weeping

Weeping is defined as the seepage or spontaneous dripping of fluid through the membrane.  Weeping leads to loss of solution/sample from the wells, resulting in reduced signal detection and/or sample recovery. Weeping is messy and can cause problems with automated instrumentation as well as contamination. The AcroPrep 96 plate is engineered to minimize weeping, permitting fluid flow only when force (e.g., vacuum, centrifugation) is applied. Table 2 shows the performance of the AcroPrep 96 filter plate with GHP membrane incubated with various commonly used solutions at room temperature. AcroPrep 96 filter plate with GHP membrane has also been tested for use at high temperature incubations. Results with water indicated no weeping from any well when incubated at 37 °C or 65 °C for 24 hours (data not shown).

Crosstalk

Crosstalk describes the situation where the contents of one well "leaks" into adjacent wells. Crosstalk can occur upstream between wells in a plate or downstream following filtration into a receiver plate. Elimination of crosstalk upstream in the filter plate using the AcroPrep 96 device is assured through intentional design features, such as individually sealed membrane disks in each well using proprietary sealing technology (Figure 1). For applications requiring filtrate collection, crosstalk can occur downstream in the receiver plate. This problem has been addressed in the AcroPrep 96 filter plate by engineering outlet frits (flow directors) optimized to minimize sputtering during filtration and ring splash guards to reduce the effects of sputtering even further. Figure 2 shows that crosstalk was 15-fold less when using AcroPrep 96 plate with GHP membrane than when using a competitor plate.

Figure 1
Crosstalk analysis of filter plate

Fluorescein dye (200 µL of a 2 µg/mL H₂O stock) was added to wells of AcroPrep 96 GHP (5 plates total) in a checkerboard pattern. Alternate wells were filled with 200 µL of water. The fluid was evacuated from each filter plate using vacuum filtration at 12 in. Hg for 15 seconds and the filter plate read in a PerkinElmer Life Sciences Wallac VICTOR* 1420 Multilabel Counter. Wells filled with water which show a CPS reading above the dashed line (5.2X10⁴ CPS, which is = "Avg" Bkgd + 5% Signal) in the graph constitutes a crosstalk event.

Figure 2
Crosstalk analysis of filter plate

Fluorescein dye (200 µL of a 2 µg/mL H₂O stock) was added to wells of AcroPrep 96 plate with GHP membrane (5 plates total) and a competitor plate in a checkerboard pattern. Alternate wells were filled with 200 µL of water. Fluid was evacuated from wells into a solid bottom receiver plate using vacuum filtration at 12 in. Hg for 15 seconds. Filter plates were carefully removed and receiver plates read in a PerkinElmer Life Sciences Wallac VICTOR 1420 Multilabel Counter.  Wells filled with water which show a CPS reading above the dashed line "Avg" Bkgd + 5% Signal) in the graphs constitute a crosstalk event. 

Solid-phase synthesis involves conducting multi-step reactions then driving them to completion by adding excess reagents and washing them away after each reaction step. Cleavage of compounds from the solid support using an optimized cleavage reaction is critical for purity and yield of the final "library" product. The AcroPrep 96 filter plate with GHP membrane allows a convenient platform to carry out all of these reactions, from wash steps to final cleavage, without the addition of interfering extractable components or cross contamination. Solid-beads can be washed with organic solvents after each synthetic process by simple filtration due to the robust compatibility of the AcroPrep 96 plate with GHP membrane. Both the polypropylene housing and the hydrophilic polypropylene membrane are well suited for this application.   This filtration ability allows the processes in combinatorial organic synthesis to be simplified with the "Wash and Remove: ease afforded by a filtrate plate. 

Step-by-step Protocol

1.  Pre-Screening and Rinsing of Plate (optional): For added insurance and maximal sample  recovery, the AcroPrep 96 filter plate with GHP membrane can be pre-screened and  pre-rinsed. To do this, pre-flush or pre-wet  each well with 100 µL of synthesis or cleavage buffer and filter through into a chemically compatible receiver plate (e.g., Matrix polypropylene). For details of filtration technique, see step 2 for manual vacuum filtration, step 3 for automated vacuum filtration, and step 4 for centrifugal filtration.

2.  Manual Vacuum Filtration: (Not recommended for extremely volatile solvents where quantification is a concern.)
2a. Place AcroPrep 96 plate on the vacuum manifold (include chemically compatible receiver to collect filtrate protect vacuum source from contamination) and apply vacuum. Most house vacuum sources do not exceed 15 in. Hg (38.1 cm Hg), however, AcroPrep 96 plate is capable of tolerating much higher vacuum pressures (30 in. Hg).
2b.  Release vacuum slowly. (Do not release vacuum by pulling the corner of the plate, as it will degrade the manifold gasket.)

3.  Automated Vacuum Filtration:
3a.  Set up vacuum manifold with gasket on platform of liquid handler. Check for proper outlet tubing and trap for the collection of liquid waste. For gasket choice, we recommend Packard Bioscience's PVM manifold gasket, which is made of 1/8 inch thick neoprene.
3b.  Place blot tray on liquid handler platform. The blot tray is used for the removal of hanging drops from the filter plate, if necessary.
3c.  At the beginning of each run, program software to direct robotic arm to place a chemically-compatible receiver plate into the vacuum manifold.
3d.  Program robotic arm to pick up AcroPrep 96 plate from a designated space on the platform and place onto coordinates corresponding to the vacuum manifold.
3e.  Program instrument to apply a high initial vacuum (up to 30 in. Hg) for 2 seconds to help seal the plate to the manifold, followed by a lower vacuum pressure to evacuate the wells. To determine evacuation time, vacuum filter test a representative solution and double the filtration time to ensure that all wells empty.
3f.  If desired samples were collected in receiver plate, have robotic arm pick up and discard the AcroPrep 96 filter plate. Then have robotic arm retrieve and process receiver plate accordingly. Some automation equipment may require disassembly of vacuum manifold prior to receiver plate retrieval. If desired samples were collected on the membranes of the filter plate, have robotic arm pick up the AcroPrep 96 filter plate and place it onto blot tray to remove any hanging droplets. Then process filter plate accordingly. 

4.  Centrifugal Filtration:
4a.  Place AcroPrep 96 filter plate on top of a chemically compatible receiver plate prior to solution addition to minimize risk of weeping with organic solvents.
4b.  Insert filter and receiver plates together into a standard swinging bucket microtiter  plate rotor assembly.  4c.  Centrifuge. As a general guideline, centrifugation at 500 x g for 1-2 minutes is sufficient to evacuate contents of well. (Speed times will vary depending on solution viscosity.)

Solid-phase extraction (SPE) techniques require the elution of bound analyte of interest typically from SPE cartridges or disks. Because most SPE cartridges or disks contain bonded silica or resin solid sorbents which can also elute off as "fines" or "particulates", a clean-up step prior to analytical or chromatographic analysis (e.g., HPLC, GC-MS, etc.) is highly recommended following solid-phase extraction.

Step-by-step Protocol

1.  Pre-Screening and Rinsing of Plate (optional): For added insurance and maximal recovery of analyte, the AcroPrep 96 plate with GHP membrane can be pre-screened and pre-rinsed. To do this, add 100 µL of elution buffer and filter through into a chemically compatible receiver plate (e.g., Matrix polypropylene). For details of filtration technique, see step 2 for manual vacuum filtration, step 3 for automated vacuum filtration, and step 4 for centrifugal filtration.

2.  Manual Vacuum Filtration: (Not recommended for extremely volatile organics where quantification is a concern.)
2a.  Place AcroPrep 96 filter plate with GHP membrane along with a chemically compatible receiver plate on the vacuum manifold.
2b.  Transfer eluates from SPE into wells of AcroPrep 96 filter plate with GHP membrane. 
2c.  Apply vacuum. Most house vacuum sources do not exceed 15 in. Hg (38.1 cm Hg); however, AcroPrep 96 is capable of tolerating much higher vacuum  pressures (30 in. Hg). 
2d.  Release vacuum slowly. (Do not release vacuum by pulling the corner of the plate, as it will degrade the manifold gasket.)
2e.  Discard AcroPrep 96 filter plate with GHP membrane and disassemble vacuum manifold to retrieve receiver plate. Contents collected in receiver plate are now particulate-free and ready for further analytical analysis.

3.  Automated Vacuum Filtration:
3a.  Set up vacuum manifold with gasket on platform of liquid handler. Check for proper outlet tubing and trap for the collection of  liquid waste. For gasket choice, we recommend Packard Bioscience's PVM manifold gasket, which is made of 1/8-inch thick neoprene.
3b.  Place blot tray on liquid handler platform (optional). The blot tray is used for removal of hanging drops from the filter plate prior to disposal in order to minimize equipment contamination.
3c.  Add to the end of the SPE program software the following steps for particulate removal using AcroPrep 96 GHP:
3i.  Direct robotic arm to place a chemically-compatible solid bottom plate along with an AcroPrep 96 filter plate on top into the vacuum manifold.
3ii.  Instruct liquid handler to transfer contents of eluates from SPE into corresponding wells of AcroPrep 96 filter plate with GHP membrane.
3iii.  Program instrument to apply a high initial vacuum (up to 30 in. Hg) for 2 seconds to help seal the plate to the manifold, followed by a lower vacuum pressure to evacuate the wells. To determine evacuation time, vacuum filter test a representative solution and double the filtration time to ensure that all wells empty.
3iv.   Have robotic arm pick up and discard the AcroPrep 96 filter plate. Optional: To minimize equipment contamination, program robotic arm to place filter plate on top of blot tray to remove hanging drops prior to filter plate disposal.
3v.  Instruct robotic arm to disassemble vacuum manifold, if necessary, and retrieve collection plate. Sample contents collected in solid bottom plate are now particulate-free and ready for further analytical or chromatographic analysis.

4.  Centrifugal Filtration:
4a.  Place an AcroPrep 96 filter plate on top of a chemically compatible receiver plate.
4b.  Transfer eluates from SPE into wells of AcroPrep 96 filter plate with GHP membrane. 
4c.  Insert filter and receiver plates together into a standard swinging bucket microtiter plate rotor assembly. 
4d.  Centrifuge. As a general guideline, centrifugation at 500 x g for 1-2 minutes is sufficient to evacuate contents of well. (Speed times will vary depending on solution viscosity.)