PROTAC Bioanalysis: Challenges and Strategies

Traditional small-molecule drugs or large molecule antibodies generally must be bound to the active sites of an enzyme or receptor to exert their therapeutic effects, but 80% of the targets in human cells lack such sites. Proteolysis-Targeting Chimera (PROTAC)*, as an emerging strategy for new small-molecule drug development, significantly combine the advantages of small-molecule compounds and small-molecule nucleic acids. They could capture target proteins through any binding site anywhere in a cell, making it easier to enter cells and exert the desired effect. PROTAC technology has embraced constant iterations and updates and has become a hot research area in recent years.

However, the mechanism involved in PROTAC’s crossing cell membrane remains unclear. Therefore, more theoretical and practical research is necessary for supporting the research on its absorption, distribution, metabolism, excretion, and toxicology. Despite PROTAC have substantial potential, their complex structures may result in poor druggability, posing huge challenges to clinical trials, bioanalysis, and PK/PD studies. This article focuses on some challenges and practical experiences in PROTAC bioanalysis. Welcome to read our series of previously published articles on PROTAC, including an overview and its DMPK research strategy, solubility and permeability, and metabolism strategies.

Key points of PROTAC bioanalysis

In terms of bioanalysis, a PROTAC molecule consists of a target protein ligand, linker, and E3 ligand, resulting in a relatively large molecular weight, typically around 1,000 Da. Multi-charged ions in mass spectrometry could cause signal dispersion, potentially leading to a loss of sensitivity. In addition, the linker structure in a PROTAC molecule is fragile and prone to breakage. Based on our practical experience, optimization of MS parameters is crucial in the LC-MS/MS analysis process to avoid in-source fragmentation. Furthermore, PROTAC compounds often have multiple chiral centers, which can result in peak splitting during liquid chromatography. Therefore, adjusting the liquid phase method to optimize peak shape is necessary for achieving smoother analysis. The exposed ions may also non-specifically bind to experiment material such as glass or plastic ¹. Hence, it is essential to eliminate non-specific adsorption to ensure no compound loss during the operation. Although the lyophilized powder of such compounds is stable, some hidden stability issues may arise when they exist in a biological matrix. Therefore, timely detection and preventive measures are necessary to ensure data accuracy. The following are the key points for PROTAC bioanalysis:

1. Optimal MS parameters: The fragile linker structure in PROTAC makes it prone to in-source fragmentation, which can be reduced by optimizing the low ionizing energy and ion source temperature. In addition, for compounds with high molecular weights, the MS conditions are further optimized to enhance sensitivity by seeking out multiple-charged. The current LLOQ (Lower Limit of Quantification) can reach 1 ng/mL.

2. Peak shape optimization- peak splitting: The existence of multiple chiral centers in the structure of PROTAC leads to peak splitting during liquid chromatography. By optimizing the method of liquid chromatography (such as mobile phase replacement, chromatographic column screening, gradient regulation, and ion source temperature reduction), a symmetrical and good peak shape can be achieved and the compound's signal can be enhanced.

 Figure 1. Comparison of Compound A Chromatographic Peaks Before and After Optimization

3. Elimination of non-specific binding: PROTAC is a small molecule compound with a large molecular weight (~1000 Da) and exposed ions tend to non-specifically bind to the surface of experiment material such as glass or plastic. Therefore, it is necessary to use appropriate container materials to collect a sample after dosing, use low-binding materials as much as possible, and investigate the non-specific binding of compounds during the development of analytical methods. If non-specific binding was found, a certain proportion of desorbent (such as Triton X-100 and Tween 20) should be added to block the non-specific binding sites, ensuring that the compound remains intact during operation (Table 1).

Table 1. Comparison of Compound B Transferred Three Times in Blank Plasma Before and After Adding a Desorbent

Note: T1: Compound B was transferred once in blank plasma; T2: Compound B was transferred twice in blank plasma; T3: Compound B was transferred three times in blank plasma

4. Controllable matrix stability: PROTAC exhibits distinctive and covert plasma stability issues. Typically, enzyme activity in the fresh matrix is higher than that in the frozen matrix, making enzyme-sensitive compounds less stable in the fresh matrix or samples. Some PROTAC compounds are stable when placed at room temperature in the fresh matrix for 2 hours, but they become highly unstable in the frozen matrix, and no inhibitors have been identified to effectively stabilize them (Table 2). Therefore, a fresh blank matrix should be used to prepare calibration standards and quality control samples.

Table 2. Comparison of Stability of Compound C in Fresh and Frozen Plasma at Room Temperature for Two Hours

Furthermore, it is necessary to avoid repetitive freezing of samples to ensure the accuracy of the unknown sample's concentration. After collecting biological samples, protein precipitation is directly carried out to obtain the preserved supernatant for testing. In this case, the stability of the treated supernatant is the main concern. In the case of compound C, a standard solution was quantitatively added to the fresh matrix to simulate the real sample. The supernatant, after protein precipitation, was placed in a sample injector at 4°C and a refrigerator at -40°C for 24 hours, respectively. Next, the supernatant of 4°C and -40°C were injected alongside the 0-hour sample, indicating that the supernatant was stable after storage in sample injector at 4°C and refrigerator at -40°C for 24 hours (Table 3). This shows that it is feasible to obtain and store the supernatant samples for preservation in the analysis of a DMPK project by immediately performing protein precipitation on biological samples after collection.

Table 3. Stability of Compound C in Supernatant of Fresh Plasma

Each step of the bioanalysis operation is interlinked. During the sample pretreatment stage, the use of HP D300e digital titration equipment allows for swift preparation of calibration standards and quality control samples within 8 seconds, eliminating the need for multiple transfers of compound solutions and mitigating non-specific binding. Biological samples are precipitated immediately after collection, and the whole sample pre-treatment process is carried out on wet ice conditions to ensure the accuracy and reliability of sample concentration detection, which provides reliable data support for the absorption, distribution, metabolism, and excretion experiments of PROTAC in vivo study.

PROTAC bioanalysis process:

Based on the different characteristics of PROTAC and the above-mentioned analysis experience, the PROTAC bioanalysis process is summarized as follows:

1. Optimization of MS parameters: Adjusting ionizing energy and ion source temperature; exploring multiple charges to improve sensitivity;

2. Optimization of liquid chromatography conditions: Screening for a suitable chromatographic column, mobile phase, and mobile phase gradient to obtain a better peak shape;

3. Elimination of non-specific binding: Carry out the method development initially and screen suitable desorbents if adsorption occurs.

4. Optimization of sample processing procedure: Rapid operation at low temperatures, precipitation immediately after sample collection, and using the fresh matrix to prepare calibration standards and quality control samples.

5. Method reproducibility: Further optimizing the method to meet analysis requirements (reproducibility, LLOQ, matrix effects, etc.).

Figure 2. WuXi AppTec DMPK Biological Sample Analysis Platform

The Non-GLP Bioanalysis team of the DMPK Services Department of WuXi AppTec has an integrated and fully capable bioanalysis platform that could provide bioanalysis services for various innovative molecular entities throughout the pre-clinical stages of drug development. The team has several years of experience in using the LC-MS/MS platform for PROTAC biological sample analysis. Moreover, the team has completed analysis on tens of thousands of biological samples, assisting customers in developing PROTAC. 

Summary and outlook

In the process of PROTAC development, the Non-GLP Bioanalysis team of the DMPK Services Department of WuXi AppTec offers a well-designed PROTAC compound analysis strategy, which

assists customers in efficiently completing experiments with accurate data and accelerating their drug discovery. We have a professional analysis team, advanced analytical instruments and equipment, comprehensive information management, and a reliable quality management system. These strengths could empower customers and efficiently complete bioanalytical services in all stages from drug screening to drug clinical trial application.

Click here to learn more about the strategies for PROTAC, or talk to a WuXi AppTec expert today to get the support you need to achieve your drug development goals.

*PROTAC® is a registered trademark of Arvinas. In this article, PROTAC specifically refers to the abbreviation of Proteolysis-Targeting Chimera as therapeutic modalities.

Authors: Tiantian Dang, Weimin Hu, Weiqun Cao, Lili Xing

Committed to accelerating drug discovery and development, we offer a full range of discovery screening, preclinical development, clinical drug metabolism, and pharmacokinetic (DMPK) platforms and services. With research facilities in the United States (New Jersey) and China (Shanghai, Suzhou, Nanjing, and Nantong), 1,000+ scientists, and over fifteen years of experience in Investigational New Drug (IND) application, our DMPK team at WuXi AppTec are serving 1,500+ global clients, and have successfully supported 1,200+ IND applications.