Aggregated Singletons for Automated Purification Workflow

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 sulfoxide solutions for biological screening. The singleton samples submitted are aggregated in a mini-array of 10–12 samples and then are analyzed 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 analogs 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) 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 sulfoxide (DMSO) solutions for biological screening. The workflow for compound synthesis to biological screening is shown in Figure 1. 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).

Figure 1: Flow chart showing the steps in the process from compound synthesis to screening.

Experimental

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) 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 Matrix-Box container.

Figure 2: Outline of the ASAP process.

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). 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 analyzed using reversed-phase 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) and a Polymer Labs 2100 ELSD system (Agilent Technologies, Santa Clara, California). 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 analyzed 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). The dry fraction tubes are then weighed on Tecan weighing stations.

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.

Results and Discussion

The ASAP process was introduced in Pfizer R&D (Groton) in April 2009. A major contributor to the success of ASAP is the strong partnership between the company's Analytical Chemistry and Medicinal Chemistry lines. A point of contact (POC) was established for each project using ASAP to facilitate communication between the two lines. Training sessions were conducted for each project joining ASAP. There was a gradual ramp-up of projects added to ASAP to ensure continuous progress of implementation.

Figure 3: Singleton compound analyzed using ASAP: (a) pre-QC analysis, (b) preparative run, and (c) final QC analysis. (a) 50 mm X 4.6 mm, 5-μm Waters XBridge C18; mobile phase A: 0.03% ammonium hydroxide in water (v/v); mobile phase B: 0.03% ammonium hydroxide in acetonitrile (v/v); gradient: 5–95% B (linear) in 4.0 min, hold at 95% B to 5.0 min; flow rate: 2.0 mL/min. (b) Column: 100 mm X 19 mm, 5-μm Waters XBridge C18; mobile phase A: 0.03% ammonium hydroxide in water (v/v); mobile phase B: 0.03% ammonium hydroxide in acetonitrile (v/v); gradient: 5–50% B (linear) in 8.0 min, 50–100% B (linear) to 8.5 min, hold at 100% B to 10.0 min; flow rate: 25 mL/min. (c) Column: 50 mm X 4.6 mm, 5-μm Waters Atlantis dC18; mobile phase A: 0.05% trifluoroacetic acid in water (v/v); mobile phase B: 0.05% trifluoroacetic acid in acetonitrile (v/v); gradient: 5–95% B (linear) in 4.0 min, hold at 95% B to 5.0 min; flow rate: 2.0mL/min.

Examples of Samples Purified by ASAP

Figures 3 and 4 show pre-QC, prep, and final QC data for samples analyzed by ASAP. Figure 3 is an example of a sample that was submitted to ASAP with an initial purity of ~15%. The target mass was isolated and enough material was delivered to the materials management department for screening. Figure 4 is an example of a complex sample. Under acidic conditions peak 4 in Figure 4a was coeluted with the target peak. However, after a method was developed in basic conditions, separation was obtained and the purified compound easily passed the final QC (Figure 4c).

Figure 4: Singleton compound analyzed using ASAP: (a) pre-QC analysis, (b) preparative run, and (c) final QC analysis. (a) Column: 50 mm X 4.6 mm, 5-μm Waters XBridge C18; mobile phase A: 0.03% ammonium hydroxide in water (v/v); mobile phase B: 0.03% ammonium hydroxide in acetonitrile (v/v); gradient: 10–95% B (linear) in 4.0 min, hold at 95% B to 5.0 min; flow rate: 2.0 mL/min. (b) Column: 100 mm X 19 mm, 5-μm Waters XBridge C18; mobile phase A: 0.03% ammonium hydroxide in water (v/v); mobile phase B: 0.03% ammonium hydroxide in acetonitrile (v/v); gradient: 10–100% B (linear) in 8.5 min, hold at 100% B to 10.0 min; flow rate: 25 mL/min. (c) 50 mm X 4.6 mm, 5-μm Waters Atlantis dC18; mobile phase A: 0.05% trifluoroacetic acid in water (v/v); mobile phase B: 0.05% trifluoroacetic acid in acetonitrile (v/v); gradient: 5–95% B in 4.0 min, hold at 95% B to 5.0 min; flow rate: 2.0 mL/min.

ASAP Workflow Deliverables and Advantages

• A high quality of purified sample is delivered to the materials management department with a 2–3 day turnaround time.

• The process provides a high level of purification expertise and technology. ASAP can purify compounds that for chemists find difficult to purify using conventional techniques.

• The process provides a high success rate (>90% samples are registered).

• Postpurification analysis: The final purity is assessed by MS–UV–ELSD and QC data are uploaded into the Global Analytical database.

• Final purity information (purity and weight recovery) is available in the research database.

• An e-mail notification is sent to the chemist. Data can be accessed through electronic laboratory notebook software.

• The process increases the likelihood of running mini-arrays to generate 5–10 analogs in a final step rather than one or two, with the same 2–3 day turnaround time.

• The ASAP process allows projects to choose synthetic routes that diverge at a late stage, which is the most effective way to prepare large sets of analogs.

• The ASAP process saves time and lets chemists focus on high-value activities such as making compounds for preliminary toxicity studies.

Conclusions

To date, more than 5700 singletons have been submitted and >90% of the samples have been successfully purified. ASAP is currently supporting approximately 25 projects across different therapeutic areas. Although the samples are purified in an automated purification mode, the versatility of the technology allows purification of diverse molecules with respect to compound solubility, polarity, and molecular weight. The next steps involve enhancing purification technologies such as adding normal-phase chromatography, supercritical fluid chromatography, and on-column solvent exchange techniques to increase the scope of available chemical space.

Acknowledgments

The authors would like to thank David Price, Rose Gonzales, and Mark Noe for sponsoring ASAP and providing resources and staffing.

Bhagyashree A. Khunte is a Principal Scientist and Laurence Philippe is an Associate Research Fellow in the Analytical Chemistry department at Pfizer Global R&D (Groton, Connecticut). Direct correspondence to Bhagyashree.Khunte@pfizer.com.