Aggregated singletons for automated purification (ASAP) is a purification workflow that provides singleton sample purification,
registration and delivery to the materials management department as 30 mM dimethyl sulphoxide solutions for biological screening.
The singleton samples submitted are aggregated in a mini-array of 10–12 samples and then are analysed using an automated purification
process. The steps in the process include pre-quality control (QC), preparative chromatography, solvent evaporation, reformat
dilution and duplication, final QC and final registration. The turnaround time from samples received to delivery to materials
management is two or three business days. The final QC data are uploaded to a database and are available to chemists via electronic
laboratory notebook software. The final purity, weight recovery and registration information is available in a research database
and an e-mail notification of completion is sent to the chemist. ASAP enables a high purification success rate; increases
the likelihood of running mini-arrays that generate 5–10 analogues in a final step rather than one or two with the same 2–3
day turnaround time; and provides expert-level service and technology.
The aggregated singletons for automated purification (ASAP) workflow was introduced at Pfizer (Groton, Connecticut, USA) in
April 2009. Singletons are unique, individual final compounds synthesized for in-vitro testing. The main driver and objective behind this initiative was to save synthesis costs and time. Typically a majority
of the singletons are made in 100–300 mg amounts. Datasets across various projects show that only ~20% of the singletons survive
primary and secondary screenings and hence ~80% of the compounds are made unnecessarily on the large scale. Pharmaceutical
chemists can save considerable time and cost by making smaller (30–50 mg) batches. The chemists can submit synthesized compounds
(30–50 mg) to ASAP to be purified, registered and delivered to the materials management department as 30 mM dimethyl sulphoxide
(DMSO) solutions for biological screening. The workflow for compound synthesis to biological screening is shown in Figure
Figure 1: Flow chart showing the steps in the process from compound synthesis to screening.
The turnaround time from samples received for purification to delivery to the materials management department is two to three
business days. Centralizing this activity in the purification group allows greater time for higher value tasks to be completed
by practicing chemists; the purification scientists can provide expert-level service and technology, and also more opportunity
for harmonization with screening (consistent and high quality samples delivered for biological assays).
Following are the steps for sample submission to ASAP: The samples submitted are final products (compounds going for biological
screening — no intermediates). The crude weight of the compound is 10–50 mg. Some type of an initial sample workup (for example,
liquid–liquid extraction, solid-phase extraction and so forth) is highly recommended to remove metals or reagents used during
the synthesis. The crude sample is fully dissolved in 900 µL of DMSO, filtered and placed in a 2D bar-coded, matrix capped
tube. The crude sample is then registered as an "in-production singleton" sample via Chemistry e-Notebook electronic laboratory
notebook software (CambridgeSoft, Cambridge, Massachusetts, USA) using the software's Global Registration tool, which has
been customized for Pfizer. The biology assay–screen panel is assigned and the sample is submitted for purification using
Pfizer's Auto-Purification submission website. The sample is then dropped off in the assigned submission dry boxes in a Singleton
The ASAP process is outlined in Figure 2. A total of 10–12 singletons are aggregated in a plate format and associated with
a bar code using a 2D Matrix tube reader (Thermo Fisher Scientific, Waltham, Massachusetts, USA). An analytical plate of fixed
concentration (1 mg/mL) is created using a Tecan Freedom Evo liquid handling system (Männedorf, Switzerland). The samples
in the analytical plate are then analysed using reversed-phase (RP) liquid chromatography–mass spectrometry (LC–MS) and evaporative
light scattering detection (ELSD) techniques. The analytical system comprised a Waters 2795 Alliance high performance liquid
chromatography (HPLC) system and ZQ mass spectrometer (Milford, Massachusetts, USA) and a Polymer Labs 2100 ELSD system (Agilent
Technologies, Santa Clara, California, USA). The initial purity of the sample can range from 5% to 80%. The pre-quality control
(QC) gradient methods are 5 min in length with 2 mL/min flow-rates. Different column chemistries, modifiers and gradient conditions
are used to develop methods best suited for purification. Even though the samples are aggregated in a miniplate and analysed
in a high-throughput mode, a method is developed for each individual sample. The best method and conditions are then transferred
to the preparative purification step. A Waters Auto-Purification Fractionlynx system is used for purification. Fraction collection
is mass triggered. The preparative methods are gradients run over 10 min. The collected fractions are evaporated using Genevac
Mega evaporators (Gardiner, New York, USA). The dry fraction tubes are then weighed on Tecan weighing stations.
Figure 2: Outline of the ASAP process.
The dried, purified material is dissolved in DMSO to make 30 mM solutions. A maximum of 900 µL of the 30 mM DMSO solution
is transferred to a plate. A daughter analytical plate is also created by transferring a 5 µL aliquot and adding enough DMSO
to result in a 0.5 mg/mL concentration. Excess 30 mM DMSO solution is transferred to bar-coded 2 mL vials. Tecan liquid handling
systems are used for dispensing the DMSO and transfer of samples to the plates and vials. The samples in the vials are evaporated
using a Genevac HT-12 evaporator in a two-step evaporation process.
The final QC is performed using the HPLC–MS–ELSD system or Waters Acquity UPLC/SQD/PL 2100 ELSD units, and the samples are
registered if they meet the purity criteria (>80% purity by UV at 215 nm, >85% purity by ELSD and >50% mass spectral purity).
Orthogonal QC methods are selected to ensure the final purity of the samples. The solubilized plate (maximum up to 900 µL
of 30 mM DMSO stock) is sent to the materials management department for assay preparations. The compound is then released
for the requested screening. The dry compounds are registered and shipped to the Pfizer Neat Store. The pre-QC and final QC
data are uploaded in Pfizer's Global Analytical database as a PDF and the file also gets linked to the chemist's e-Notebook
submission page. The recovery amount, purity and registration data are available through Pfizer's Research database. An e-mail
notification is sent to the chemists to inform them that the samples have been delivered to the materials management department.
A results spreadsheet is attached that provides detailed information regarding the samples, including the QC gradient conditions,
mass observed, retention time, purity data and total recovery after purification. The entire process and data flow are handled
through customized software. The software has been specifically designed for automated batch-singleton and library purification
workflow. The software enables a user to handle multiple plates with accurate data flow. It also provides structures, chemical
properties, CLogD data and acid–base labile prediction information for the compounds associated in the plate. This information
helps minimize the time required for method development.