How can Automation Accelerate In Vitro Absorption, Distribution, Metabolism, and Excretion Studies?

In vitro pharmacokinetic (PK) studies are characterized by high throughput, high precision, and high efficiency. Compared to traditional manual assays, automated liquid handling workstations can precisely and efficiently complete tasks such as sample addition, gradient dilution, and liquid distribution, effectively reducing human errors, and improving the speed, quality, and throughput of in vitro absorption, distribution, metabolism, and excretion (ADME) testing.

The in vitro ADME team of the DMPK Service Department, also known as "in vitro ADME," studies the compounds’ PK properties, such as absorption, distribution, metabolism, and drug–drug interactions, using in vitro assays. Following the introduction of the first automated liquid handling workstation in 2014, the in vitro ADME team has continuously expanded the platform and upgraded the automation level of the workstation to improve work efficiency and the quality of research data.

Currently, an automation platform with multiple high-throughput integrated liquid handling workstations that are well aligned with the in vitro ADME business has been established. It covers various types of in vitro ADME assays and efficiently supports new drug development projects.

Advantages of High-throughput Integrated Automation Platform

1.High-throughput

High-throughput integrated liquid handling workstations come standard with 96-channel pipetting heads, which allows the processing of entire 96-well plates in a single run. The workstations can also be upgraded to 384-channel pipetting heads. Routine laboratory operations such as substrate, compound, and termination solution addition can be quickly performed. The workstation typically has approximately 50 plate positions (expandable to accommodate additional positions), which enables batch processing of multiple 96-well plates or multiple assay rounds. The liquid handling speed of the workstation is fast, considerably improving throughput.

Figure 1. Table top and 96-channel pipette head of the high-throughput integrated liquid handling workstation

2.High precision

The High-Throughput Integrated Liquid Workstation allows accurate pipetting of small volumes (0.5 μl) and the setting of appropriate liquid parameters according to the type of liquid to ensure accurate pipetting. Different pipetting speeds and air column volumes can be set according to the characteristics of the liquid, such as viscosity, to ensure accurate pipetting. As a result, the experimental results performed by the workstation are highly reliable and reproducible. For example, when applied to the configuration of standard curves, the accuracy (Accuracy (%)) is about 100 (Fig. 2), greatly exceeding the accuracy acceptance standard of 80 ≤ Accuracy (%) ≤ 120.

Figure 2. Accuracy of high-throughput integrated liquid workstations for preparing standard curves

3.Fast speed

High-throughput integrated liquid handling workstations operate at a fast speed, allowing projects to be completed efficiently. The workstations can extend the 8-h working limit of instruments and automatically perform assays overnight, speeding up project progress and improving the efficiency of the in vitro ADME team. Specifically, the efficiency value has increased by more than double compared to that before the introduction of high-throughput integrated liquid handling workstations. Furthermore, the high-throughput workstation ensures project timelines are maintained with no waiting time for IND project initiation.

4.High success rate

Before officially launching the operational workstation, each step of the experimental procedure was validated and optimized numerous times to ensure a smooth flow and compliance of the data with acceptance criteria. The workstation executes projects automatically using preset standardized procedures, effectively avoiding human error and enhancing the success rate of assays.

 Figure 3. Advantages of automated liquid handling workstations

Upgrade of Developed In Vitro ADME Automation Platform

To continuously improve its delivery efficiency and quality, the in vitro ADME automation platform is continuously evolving. From small semi-automated to stand-alone automated liquid workstations followed by high throughput integrated liquid workstations, the platform has now evolved to version 3.0 (see Figure 4).

Figure 4. Upgrade of developed in vitro ADME automation platform

Version 1.0:

The liquid handling operations of the assays in this version were performed using a small semi-automatic liquid workstation with low throughput. Consequently, this workstation required frequent manual operation, resulting in low overall efficiency.

Version 2.0:

Automation workstations that could handle liquids in batches were introduced in this version and then automated standard solutions were developed for each type of assay. Subsequently, "instruments were introduced to facilitate assay performance," which increased not only productivity but also efficiency. The introduction of automated workstations has considerably increased assay throughput and improved operational efficiency.

Version 3.0:

The completion of assays by decentralized individual liquid handling workstations still required numerous manual steps in this version, and the efficiency was not substantially improved. Based on our business model, we built a high-throughput integrated liquid handling workstation with an automated pipetting workstation as the core and peripheral integrated functional equipment. The automation functions of the workstation were expanded beyond automated liquid handling and plate transfer and are controlled by the software incorporated. Compared to a single workstation, this automation platform can perform functions other than liquid handling, including constant temperature and uniform oscillation incubation; centrifugation and solid-liquid separation, sealing and peeling, and cryopreservation of sample plates. Presently, we have achieved automation of the whole process with the continuous execution of multiple assay sets. In the future, we will customize and integrate more diverse equipment to further enhance the high-throughput advantages of automation.

Currently, the full-process automation solutions for various types of in vitro ADME assays have been developed and validated, demonstrating the feasibility and accuracy of conducting assays using automated instruments. Some validation data, including microsome metabolic stability (MMS) validation, plasma protein binding (PPB) validation, and cytochrome P450 (CYP) inhibition (DDIM) validation for assays are shown in Figure 5.

Figure 5. Validation results of standardized full-process automation solutions for selected in vitro assays

Extending the 8-h working system of the instrument: The integrated liquid handling workstation can continuously perform full-process assays. We have validated the procedures for full-process assays multiple times and adjusted and optimized them to improve error tolerance. The improvements include automatic correction of sample plate positioning to avoid instrument errors caused by improper placement of sample plates. Continuous adjustments and optimizations ensure the stable operation of the entire process, enabling the performance of unattended overnight reactions. Sample plates and other consumables can be arranged, and then the program can be started and left to run. The workstation can run automatically overnight, continuously conducting assays and efficiently assisting project progress.

Professional platform management: The in vitro ADME automation platform team has extensive experience and automation technicians can upgrade and advance the automation platform according to business needs. The team has also established a digital and visualized platform for operation management and the use of automation instruments. These processes allow resource configuration and platform management across multiple sites, thereby continuously improving efficiency. The management platform enables multi-user, -program, -instrument, and -site collaborative management, thereby achieving reasonable and efficient deployment, ensuring project timelines, and facilitating project implementation.

 Figure 6. Diagram of the automated workstation management platform

Outlook

WuXi AppTec DMPK has laid a solid foundation for automation and established a high-throughput integrated automation platform for in vitro ADME, enhancing delivery efficiency and quality. In the future, we will focus on building an automation laboratory that is highly aligned with the business to facilitate efficient drug discovery and development.

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

References:

[1] R. Scott Obach. Prediction of human clearance of twenty-nine drugs from hepatic microsomal intrinsic clearance data: an examination of in vitro half-life approach and nonspecific binding

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[2] Zamek-Gliszczynski MJ et. al. Validation of 96-well equilibrium dialysis with non radiolabeled drug for definitive measurement of protein binding and application to clinical development of

highly-bound drugs. J Pharm Sci. 2011 Jun;100 (6):2498507.

[3] Kye Sook Lee and Sang Kyum Kim. Direct and metabolism-dependent cytochrome P450 inhibition assays for evaluating drug–drug interactions. J. Appl. Toxicol., 2013, 33: 100-108.

Author: Binbin Qian, Xiangling Wang, Genfu Chen

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.