Degradation Products of ARV-825 Proteolysis Targeting Chimera (PROTAC) Compound: Isolation and Purification Using Analytical Scale Fraction Collection

Proteolysis targeting chimeras (PROTACs) are new drug modalities applied for the treatment of various cancer-related diseases. Since their initial discovery, numerous PROTAC molecules were designed to target a wide range of diseased proteins. The ARV-825 is a novel PROTAC compound. In this application note, degradation products of ARV-825 generated through acid hydrolysis were investigated. The degradation products were isolated using Waters™ Fraction Manager - Analytical (WFM-A) configured with an Arc™ Premier System, 2998 PDA Detector, and ACQUITY™ QDa™ II Detector. The specificity of the mass-directed trigger enabled efficient fraction collection of the target degradation products, while the time-based trigger allowed collection of closely eluting peaks. The MassLynx™ Software with FractionLynx™ Application Manager automated the collection process, while tracking the location of the collected fractions.

Benefits

• The Waters Fraction Manager - Analytical (WFM-A) configured with an Arc Premier System, 2998 PDA Detector, and ACQUITY QDa II Detector enables fast and easy collection of the target compounds.
• The low dispersion system configuration together with the use of sub-5 µm particle-size analytical columns is designed to collect narrow peaks, delivering fast chromatography with enhanced resolution for fraction collection and in an off-collection analysis mode.
• MassLynx Software with the FractionLynx Application Manager provides automated fraction collection, while tracking the location of the collected fractions.

PROTACs offer novel approaches for the treatment of cancer and other diseases through selective degradation of target proteins.^1,2 These heterobifunctional molecules contain E3 ubiquitin ligase ligand connected via a chemical linker to a ligand that recognizes and binds to the target protein. This configuration enables PROTACs to form a stable ternary complex, leading to ubiquitination and degradation of the target protein. The ARV-825 is a newly developed inhibitor using PROTAC technology that has shown effectiveness for the treatment of pancreatic cancer, melanoma, cholangiocarcinoma, thyroid carcinoma, and acute myeloid leukemia.^3-5 The ARV-825 induces degradation of bromodomain-containing protein 4 (BRD4), a target protein marker in many cancer types. Currently, little work has been reported in the literature related to the stability and impurity characterization of PROTAC drug molecules.

The forced degradation studies, also referred to as stress testing, are designed to generate degradation products or impurities that could potentially form during storage and use of drug substances and drug products. The studies provide data to support identification of possible degradants, degradation pathways and the development of stability-indicating methods.⁶ While numerous techniques can be employed to identify the unknown degradants, an analytical-scale fraction collection approach isolates a pure target compound for further characterization and identification. The WFM-A is a low dispersion fraction collector for analytical scale isolation and purification when only small amounts of sample are available.⁷ Multiple modes of fraction collection are available with WFM-A, based on the elution time, peak threshold, slope, or combinations of these parameters. When combined with mass detection, target compounds can be isolated based on a mass-directed trigger.

Work in this application note investigates degradation products of the ARV-825 molecule generated through a forced degradation study with a 0.5 M hydrochloric (HCl) acid. The degradation products were isolated and collected by using the WFM-A configured with an Arc Premier System. Using the ACQUITY QDa II Mass Detector, mass-directed fraction collection was applied to isolate the target degradation products. To verify purity, the collected fractions were analyzed using orthogonal chemistry. Time-based mode and focused gradients were employed to effectively separate and identify closely eluting components. The FractionLynx Application Manager Software facilitated the collection process.

Mass spectrometry grade solvents and formic acid were purchased from Sigma-Aldrich. ARV-825 PROTAC compound was obtained from Sigma-Aldrich.

Sample Description

Forced Degradation Sample

The ARV-825 compound was dissolved in a 45:55 acetonitrile/water diluent at 1 mg/mL. The forced degradation sample was prepared by adding 100 µL of 0.5 M HCl to the ARV-825 sample solution. The forced degradation solution was stored at room temperature for 2 hours and subsequently neutralized with 100 µL of 0.5 M of sodium hydroxide (NaOH). 

The forced degradation study of an ARV-825 compound was performed based on a previously published application note.⁸ Degradation with 0.5 M HCl for two hours at room temperature produced several degradation products. The chromatographic separation was performed on the XSelect Premier CSH C₁₈ Column using an Arc Premier System with WFM-A in injection mode only, collection off (Figure 1). The major degradation products were successfully separated, allowing for effective collection of high purity fractions. The mass-directed and time-based fraction collection triggers were applied to isolate the degradation products of ARV-825.

Mass-Directed Fraction Collection

In mass-directed mode, fractions were collected based on the molecular mass acquired on an ACQUITY QDa II Mass Detector. The ISM was used to split and dilute the flow entering the ACQUITY QDa II Mass Detector. The ISM makeup (dilution) solvent was added post-column and mixed with the flow entering the source, allowing for the addition of a modifier to enhance ionization.

In this study, various ISM makeup solvents were screened to enhance the MS signal for the degradation products. The makeup solvents included different compositions of acetonitrile and methanol in water, all containing 0.1% formic acid (Figure 2). The makeup solvent with 50:50 acetonitrile/water with 0.1% formic acid produced a robust signal for the degradation products.

A mass-directed fraction collection was applied to isolate degradation products of ARV-825. Using the target masses (m/z 640, 472, 428, and 416), fractions were successfully collected and multiple fractions pooled or combined into one vial (Figure 3). Pooling fractions allowed for collection of larger volumes. FractionLynx automated fraction collection and tracked vial location of the collected samples.

Analysis of the Collected Fractions

The fractions collected by mass-trigger were analyzed using orthogonal chemistry with XSelect HSS™ T3 Column (4.6 x 100 mm, 2.5 µm particle size, p/n 1860061510). The analysis was run on an Arc Premier System with Quaternary Solvent Manager (QSM) integrated with 2998 PDA Detector and ACQUITY QDa II Detector, controlled with Empower™ Chromatography Data System (CDS). To demonstrate purity of the collected fractions, both chromatographic and spectral purity of the degradation products were evaluated.

Analysis of fraction 1 with m/z 640 showed chromatographic purity or percentage area of 97.8% and 97.7% for single and pooled fractions, respectively (Figure 4). Both the UV and MS spectral data demonstrated spectral purity of the collected fractions, indicating target compounds are not coeluting with other components from the sample.

Fraction 2 was collected using a target mass of m/z 472. The orthogonal analysis revealed the presence of an additional peak with m/z 463 (Figure 5). Additionally, the concentration of this additional peak increased with time, as shown for the collected fraction reanalyzed a week later (Figure 5B). This indicated degradation of the target compound in the collected fraction solution.

Time-based Fraction Collection

Time-based fraction collection over a defined chromatographic region allows collection of closely eluting peaks through slicing into smaller sections.

The time-based collection mode was applied to fraction the chromatographic region near the ARV-825 peak into smaller sections (Figure 6). In this case, the collected fractions were re-injected on the WFM-A System with the same column using a focused gradient to produce a fast and efficient separation (Figure 7). Analysis of fractions 2 and 3 on an ACQUITY QDa II Mass Detector showed the presence of a peak with masses corresponding to m/z 923 and 462, specific to the ARV-825 compound.

Isolation of the degradation products of the ARV-825 compound was successfully performed using the WFM-A integrated with an Arc Premier System. Combination with the ACQUITY QDa II Mass Detector facilitated the use of a mass-directed trigger, enabling efficient fraction collection of the target degradation products. The time-based fraction collection combined with the low peak dispersion design of the WFM-A, allowed precise collection of closely eluting peaks. Purity of the collected fractions was verified through analysis using an orthogonal chemistry. MassLynx Software with FractionLynx Application Manager facilitated the fraction collection process. The WFM-A configured with an Arc Premier System enabled fast and easy collection of the target compounds in PROTACs drug molecule.

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