Drug development is a rapidly evolving field in which precision and efficiency in analysis methods are paramount. Understanding plasma protein binding (PPB) is essential for assessing drug distribution and safety in pharmaceutical research. Though reliable for certain molecular types, traditional methods of assessing PPB in oligonucleotides are challenging due to limited accuracy, low efficiency, and the high cost associated with these molecules’ unique structures, sizes, and charges. The emergence of a rapid agarose gel Electrophoretic Mobility Shift Assay (EMSA)— using a Thermo E-Gel Power Snap Electrophoresis device—offers a new approach to these challenges.
While not revolutionary, this method marks a significant step in oligonucleotide research. It simplifies the separation of free oligonucleotides from those bound to plasma proteins, integrating pre-stained gels for quicker observation and semi-quantitative assessment. This technique - noteworthy for its simplicity and potential cost-effectiveness - could enhance early-stage drug development processes. However, its current limitations and the need for further optimization underscore that while promising, it remains a method in need of refinement.
Background
Plasma protein binding is crucial to understanding the pharmacokinetic (PK) and pharmacodynamic (PD) properties of therapeutic oligonucleotides. The extent of binding to plasma proteins can significantly influence a drug's distribution, efficacy, and safety. Traditional methods for assessing PPB, including ultrafiltration and previous versions of EMSA, have faced challenges regarding accuracy, efficiency, and cost. However, the emerging rapid agarose gel EMSA method - using a Thermo E-Gel Power Snap Electrophoresis device - marks a significant step forward.
This method enables the separation of free oligonucleotides from those bound to plasma proteins in diluted plasma. This is achieved through agarose gel electrophoresis, where free nucleic acids migrate faster than their protein-bound counterparts. Crucially, this method employs pre-stained gels, facilitating rapid separation and real-time observation of the process. The values of gel bands are calculated using ImageJ software, allowing for a semi-quantitative assessment of free nucleic acids in total plasma nucleic acids.
The rapid agarose gel EMSA addresses several limitations of previous methods. Traditional EMSA methods suffer from long separation times and low throughput. They also face challenges with quantifying binding rates and lack a linear relationship in nucleic acid staining across a wide concentration range. Notably, the previously reported method failed to account for plasma dilution. Meanwhile, while ultrafiltration is accurate, it struggles with non-specific adsorption and is limited because of the characteristics of the ultrafiltration tube membrane. The complexity and low throughput of ultrafiltration, coupled with the high costs associated with LC-MS/MS (i.e., liquid chromatography-tandem mass spectrometry) measurement, makes it less than ideal for large-scale or rapid testing.
The rapid agarose gel EMSA method stands out for its efficiency, simplicity, and cost-effectiveness, addressing a critical need in drug development and pharmaceutical outsourcing. Offering a method that is quick, easy to use, and less expensive paves the way for more accessible and frequent PPB testing in the early stages of drug development. This potential could drive better-informed decisions and potentially faster progression from the screening phase to clinical trials.
Methodology
The methodology used in this study was designed to address specific challenges in pharmaceutical research. The oligonucleotides chosen for this study were based on a strategic criterion: antisense oligonucleotides (ASOs) and small interfering RNA (siRNA) drugs that are already approved by the U.S. Food and Drug Administration (FDA). This selection was pivotal for two reasons:
- These oligonucleotides have pre-existing PPB data reported in the literature, providing a foundation for comparing and validating the new method.
- Their relevance in pharmaceutical research is well-established, as they represent therapeutic agents in use or development, ensuring the practical applicability of the study's findings.
Notably, some oligonucleotides, like phosphonodiamidite morpholino oligomers (PMO)-based ASOs, were excluded due to their charge-neutral nature, making them unsuitable for electrophoresis. This exclusion criterion underscores the importance of molecular characteristics in determining the suitability of oligonucleotides for analysis using EMSA.
EMSA Methodology: Set Up and Findings
Using a Thermo E-Gel Power Snap Electrophoresis device enabled rapid separation of oligonucleotides. The process involved using agarose gel electrophoresis to separate free oligonucleotides from those bound to plasma proteins in diluted plasma. Based on their binding status, nucleic acids display different migration patterns on the gel—i.e., free nucleic acids move faster compared to the slower migration of protein-bound nucleic acids.
A critical aspect of this methodology was using pre-stained gels, which allowed for immediate visual observation and rapid separation. ImageJ software calculated the numbers of gel bands in various shades of gray, enabling a semi-quantitative assessment of the proportion of free nucleic acids. Choosing this rapid agarose gel EMSA method significantly influenced the study outcomes, particularly regarding the plasma protein binding values observed.
It's also important to acknowledge that, although innovative and efficient, this method exhibited more variability compared to the UF-LC-MS/MS (i.e., ultrafiltration-liquid chromatography-tandem mass spectrometry) method. Factors such as the quality of the gels, temperature control during the EMSA process, and the type of buffer used were identified as critical variables that could affect the results.
Despite these potential variabilities, under optimal conditions, the EMSA method demonstrated the capability to provide reliable results comparable to those obtained from ultrafiltration. This aspect highlights the importance of stringent quality control and precise experimental conditions in ensuring the accuracy and reliability of the EMSA method.
This methodology was carefully crafted to explore the potential of rapid agarose gel EMSA in PPB analysis and provide directly applicable and highly relevant insights to current pharmaceutical research. The strategic selection of oligonucleotides, coupled with the meticulous setup of the EMSA and an understanding of the method's influence on study outcomes, all contribute to the robustness and relevance of this research.
The study's findings present significant implications for drug development. Efficiency is a standout feature of the rapid agarose gel EMSA. Compared to the more complex and time-consuming UF-LC/MS-MS method, the rapid agarose gel EMSA offers a simpler and faster operation. The oligonucleotide and protein binding range can be observed in less than 10 minutes after sample loading. This capability to process several gels in a day enhances the throughput, making it highly efficient for screening large numbers of samples in the early stages of drug development.
The rapid agarose gel EMSA method also demonstrated relatively accurate results at a qualitative level, especially in the early stages of research. This is substantiated by the data of antisense oligonucleotide (ASO) compounds and various types of oligonucleotides from internal projects. In some cases, the data obtained from the EMSA method closely mirrored those from the ultrafiltration method, demonstrating its reliability.
Limitations of Traditional Methods
The rapid agarose gel EMSA method offers a more efficient and potentially more accessible approach for determining PPB in oligonucleotides. This will ultimately aid in more efficient progression of therapeutic oligonucleotides from the laboratory to clinical use. This is particularly significant considering the limitations of traditional methods, including:
Calculating PPB values
A notable finding in the study was the different plasma protein binding values for the U.S. FDA-approved GalNac-siRNAs compared to previously reported data and in-house data obtained by the UF (i.e., 50KD-ultrafiltration) method. Initially, these discrepancies were not fully understood. However, subsequent studies revealed that the fraction unbound values are highly sensitive to the buffer used in the rapid agarose gel EMSA method. Thus, selecting an appropriate buffer in future studies is crucial to obtain data comparable to other methods. For researchers, this highlights the critical role of experimental conditions in obtaining accurate PPB data, which is essential for understanding the oligonucleotides' PK properties.
Optimizing binding rate methods
While there were no groundbreaking innovations in the electrophoresis technology itself, significant improvements were made in optimizing the experimental scheme and the calculation method of the binding rate. These enhancements make the rapid agarose gel EMSA method more convenient, faster, and reliable than traditional plasma binding rate methods. The method's suitability for drug development lies in its ability to rapidly screen the PPB of oligonucleotides in the early phases of drug discovery, providing valuable data that can guide further development and optimization of therapeutic agents.
Accuracy and cost
The rapid agarose gel EMSA and the traditional UF method differ significantly in their approach to measuring PPB. While the EMSA uses gel electrophoresis for separation and conducts semi-quantitative analysis through grayscale values of stained bands, the UF method relies on ultrafiltration technology followed by LC-MS/MS for quantitative analysis. The UF method's accuracy can be impacted by factors like adsorption in the ultrafiltration tube, membrane permeability, and protein leakage rate. Moreover, the high cost of analysis and the use of organic reagents in the UF method pose additional challenges, including environmental concerns.
Future Implications of the Rapid Agarose Gel EMSA Method
Developing and successfully implementing the rapid agarose gel EMSA method to analyze plasma protein binding (PPB) of oligonucleotides has significant implications for future research. This method is anticipated to be crucial in advancing oligonucleotide research, particularly in the early stages of chemical modification.
Understanding the PPB of oligonucleotides is essential, as it influences tissue distribution and renal clearance of these molecules. The rapid agarose gel EMSA method provides a simpler, faster, and more cost-effective approach to studying PPB compared to traditional methods. Its ease of use and lower cost make it an attractive option for widespread adoption in oligonucleotide research, especially during the early phases of drug development, where rapid and frequent testing is beneficial.
Furthermore, the ability to assess the binding properties of oligonucleotides quickly and efficiently with plasma proteins can accelerate the optimization of chemical modifications and lead to more effective and safer therapeutic agents. Thus, this method can potentially streamline the development process of oligonucleotide-based drugs from discovery to preclinical stages.
Despite its advantages, the rapid agarose gel EMSA method is highly sensitive to buffer compositions and salt concentrations.
Therefore, future research should focus on optimizing experimental conditions to closely mimic physiological conditions. Achieving this would enhance the relevance and applicability of the data obtained, ensuring that the PPB values are reflective of in vivo scenarios.
In addition, identifying the main causes of the large variations observed between experiments is another critical area for further investigation. Understanding and mitigating these variations will improve the method's reliability and accuracy, making it a more robust tool for PPB analysis.
Conclusion
The rapid agarose gel EMSA method represents a noteworthy advancement in oligonucleotide research, particularly PPB analysis. Its efficiency, cost-effectiveness, and relative accuracy make it a valuable tool for early-stage drug development. While it introduces a more accessible approach for PPB assessment, its sensitivity to experimental conditions like buffer composition highlights areas for further refinement. Future research aimed at optimizing and standardizing the method will enhance its reliability and applicability. Ultimately, the rapid agarose gel EMSA method is poised to streamline oligonucleotide drug development, contributing significantly to discovering and optimizing safe and effective therapies.
About WuXi AppTec
As a global company with operations across Asia, Europe, and North America, WuXi AppTec provides a broad portfolio of R&D and manufacturing services that enable the pharmaceutical and healthcare industry around the world to advance discoveries and deliver groundbreaking treatments to patients.
Through its unique business models, WuXi AppTec’s integrated, end-to-end services include chemistry drug CRDMO (Contract Research, Development, and Manufacturing Organization), biology discovery, preclinical testing and clinical research services, and cell and gene therapies CTDMO (Contract Testing, Development, and Manufacturing Organization), helping customers improve the productivity of advancing healthcare products through cost-effective and efficient solutions. WuXi AppTec received AA ESG rating from MSCI in 2023 and its open-access platform is enabling more than 6,000 customers from over 30 countries to improve the health of those in need – and to realize the vision that “every drug can be made and every disease can be treated.
Author Details
Dr. Jie Wang - Principal Scientist, DMPK Service Department, WuXi AppTec.
Dr. Jie Wang received her Ph.D. in pharmaceutical analysis from Shenyang Pharmaceutical University in 2014. She has 10+ years of experience in preclinical DMPK. Currently, she is a principal scientist in the DMPK Service Department at WuXi AppTec. She is focusing on in vitro ADME with expertise in protein binding and drug metabolic stability assays.
Publication Detail
This article appeared in Pharmaceutical Outsourcing:Vol. 25, No. 1Jan/Feb/Mar 2024Pages: 36-38
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