Liquid biopsy analysis has emerged as a groundbreaking tool in precision medicine, offering a minimally invasive way to detect and monitor diseases through biomarkers found in body fluids such as blood, urine, saliva, and cerebrospinal fluid. Unlike traditional tissue biopsies, which require surgical extraction, liquid biopsies are non-invasive and rely on detecting components released by cells into the bloodstream or other fluids. These components, also known as biomarkers, include circulating tumour DNA (ctDNA), circulating tumor cells (CTCs), RNA, proteins, and extracellular vesicles (EVs). Among these, EVs have garnered significant attention due to their role in intercellular communication and disease progression. However, isolating high-quality EVs from complex biological fluids presents several challenges. This blog explores these challenges and introduces the Izon qEV system as a robust solution to address these challenges.

Challenges in Isolating Extracellular Vesicles (EVs) from Liquid Biopsies

Isolating EVs from liquid biopsies is a promising yet challenging process due to several factors:

1. Complexity of Biological Fluids

Biological fluids like plasma, urine, breast milk, and saliva contain a diverse mix of proteins, lipids, nucleic acids, and other particles. Plasma, for example, is highly complex, consisting of soluble proteins like albumin and lipoproteins such as high-density lipoproteins (HDL), low-density lipoproteins (LDL), intermediate-density lipoproteins (IDL), very low-density lipoproteins (VLDL), and chylomicrons. These lipoproteins share several overlapping characteristics with EVs, including size, density, and lipid content, making it difficult to separate EVs from contaminants.

Techniques like ultracentrifugation (UC) and precipitation are prone to non-specific binding, resulting in the co-isolation of contaminants like lipoproteins, protein aggregates, and free nucleic acids. UC, which uses gravitational force to pellet EVs, often results in the co-purification of particles that share similar physical characteristics with EVs, reducing the specificity of the isolation process. Precipitation-based methods, such as those using polyethylene glycol (PEG), also face similar challenges, as these polymers aggregate both EVs and non-EV components, contaminating the sample and complicating downstream analyses.

Size exclusion chromatography (SEC) SEC employs a column filled with porous beads that separates particles based on size by gravity. Smaller particles pass through the column more slowly, while larger particles (such as EVs) are excluded from the pores and travel faster. This separation results in a collection of EVs that are enriched in size while minimising contamination from smaller proteins, nucleic acids, and lipoproteins. Hence, SEC stands out for its ability to achieve high purity, reproducibility, and a gentle isolation process that preserves the integrity of EVs—making it an ideal choice for liquid biopsy analysis.

2. Heterogeneity of EVs

EVs vary significantly in size, ranging from 30 to 5000 nanometers, and include exosomes, microvesicles, and other vesicular structures, each with distinct biological roles. The lack of clear delineation between EV subtypes and the biological variability originating from different cell types and tissues complicates both isolation and standardisation. Variations in protein, lipid, and nucleic acid profiles, as well as physical properties such as density and charge, make EV isolation from different biological fluids and clinical conditions even more complex.

One of the primary advantages of the Izon qEV platform is its ability to achieve exceptional purity in EV isolation. The qEV Gen 2 Series (an improved version of the now phased-out Legacy Series) can remove 99.4% of protein, 99.9% of ApoA1 (HDL), and 99% of ApoB (IDL, LDL, and VLDL) from the plasma. These high removal efficiencies ensure that the EV sample is highly enriched and free from contaminants, which is critical for accurate downstream analyses such as proteomics, transcriptomics, and functional assays. The ability to isolate EVs with such high purity not only enhances the reliability of research findings but also makes the qEV platform a superior option for liquid biopsy applications.

3. Technical Limitations On Reproducibility & Scalability

Several technical limitations impact the efficiency, scalability, and standardisation of EV isolation. Many methods, such as UC and precipitation, suffer from low recovery rates and yield EVs of variable purity. This variability can compromise downstream applications like omics studies or functional assays. Additionally, UC is labour-intensive, time-consuming, and requires expensive, specialised equipment, making it impractical for routine clinical use. Newer methods, such as microfluidics or automated systems, offer improved scalability but are not yet widely adopted due to high costs and the need for further validation. Moreover, the lack of standardised protocols across laboratories leads to inconsistency in EV yields, purity, and integrity, making reproducibility a significant challenge to ensure consistency and reliability across research and clinical applications.

Scalability and reproducibility is essential for advancing EV research especially as demand for large-scale EV isolation grows in both research and clinical settings. However, EV isolation techniques can significantly impact the reproducibility and scalability of results.

UC, a commonly used isolation method, is known for poor reproducibility across different laboratories due to variability in ultracentrifuge models, rotors, and operator handling. These factors contribute to inconsistent EV yields and quality, with differences in sample viscosity and protocol variations further complicating the process. As a result, UC often lacks the consistency needed for scalable, reliable EV isolation.

The Izon qEV platform addresses these challenges through its standardised approach to SEC. A key feature in this system is the Automatic Fraction Collector (AFC), which plays a critical role in enhancing both reproducibility and scalability. By automating the collection of purified EV fractions and precisely weighing them as they are collected, the AFC ensures that purified collection volumes (PCV) remain consistent across isolations. This standardisation reduces user-introduced variation, such as differences in the volume of each fraction, which could otherwise introduce inconsistencies and compromise results.

Additionally, the AFC allows users to program the PCV according to their specific sample type or downstream application needs. This flexibility enhances scalability by ensuring that EV isolation can be adjusted to accommodate various sample volumes and research requirements, making it suitable for both small-scale studies and high-throughput applications.

Together, the qEV columns and AFC system provide exceptional scalability and reproducibility, enabling researchers to conduct EV isolation consistently across samples, users, and laboratories. This combination ensures reliable, comparable results that are crucial for progressing research and translating findings into clinical applications.

4. Impact on EV Integrity

Maintaining the integrity of EVs during isolation is crucial, as certain techniques may damage or alter their structure. For instance, shear forces from UC can fragment EVs, disrupting surface proteins, lipids, and RNA, which may lead to unreliable results. Chemical alterations from precipitation methods, which rely on reagents like PEG, can also leave residues that interfere with subsequent analyses, such as proteomics or RNA sequencing. These changes can compromise the biological activity of EVs, particularly in therapeutic applications where functional properties are key.

The gentle nature of the Izon qEV platform is another key advantage. Unlike methods like UC, which apply high forces that can damage or fragment EVs, SEC separates particles by gravity without the use of aggressive forces. This results in minimal shear stress and ensures that the EVs retain their structural and functional integrity. Surface proteins, lipids, and RNA content are preserved, which is crucial for ensuring that the EVs are biologically active and ready for downstream analyses. This gentle approach is especially important when isolating EVs for therapeutic applications, where maintaining the biological properties of EVs is essential for their effectiveness.

5. Clinical and Regulatory Challenges

Lastly, the clinical application of EV-based technologies faces significant challenges in reproducibility and regulatory compliance. Biological fluid variability among patients—such as differences in sample composition and EV abundance—makes achieving consistent isolation results difficult. A lack of standardised protocols often leads to inconsistent yields, purity, and integrity of EVs, hindering their use in diagnostics and therapeutics. Additionally, regulatory hurdles complicate the clinical translation of EV isolation methods. Agencies require extensive validation to demonstrate the safety, efficacy, and reliability of EV-based diagnostic tools or therapies, which involves ensuring scalability, reproducibility, and clear quality control criteria. The absence of universally accepted benchmarks for EV characterisation and functionality further adds to the regulatory complexity.

Overcoming these challenges necessitates robust and reliable EV isolation techniques. A crucial aspect is establishing standardized protocols and automated operation, to ensure consistent EV isolation across different laboratories and experiments for reliable downstream analyses and clinical translation.

Spotlight on Izon’s EV Isolation Methods & Results: Comparative Insights from Research

1. Comparing Membrane Affinity and SEC for Exosome Isolation from Plasma

The method you choose for isolating EVs can profoundly influence the purity, quality, and utility of your samples. A 2018 study by Stranska et al., published in the Journal of Translational Medicine, provides a comprehensive comparison of SEC using qEV columns versus the exoEasy membrane affinity-based kit for isolating exosome-like vesicles (ELVs) from human plasma.

Key Insights from the Study

1. Superior Purity and Specificity

qEV columns demonstrated exceptional performance in separating EVs from contaminating plasma proteins and lipoproteins. The resulting EV-rich fractions exhibited minimal impurities, a critical factor for downstream applications. In contrast, the exoEasy kit showed a high protein content and low particle-to-protein ratio, with exosome-associated markers either undetectable or present at low levels. These findings suggest that exoEasy isolates are often contaminated with non-EV plasma proteins and lipoproteins, reducing sample purity.

• Preservation of Exosome-Associated Proteins
  qEV columns retained critical exosome-associated proteins, such as syntenin-1, TSG101, and CD81, which are essential for molecular characterisation and biomarker discovery. The exoEasy kit, however, failed to enrich these markers effectively, compromising the utility of its isolates for proteomic analysis.

• Reproducibility and Speed
  SEC with qEV columns offers reproducible results and a relatively short processing time (~20 minutes), with minimal preparation effort. Although the exoEasy kit also offers a streamlined workflow, its inferior purity limits its practical utility in high-stakes applications like biomarker discovery.

• Reduced Lipoprotein Contamination
  While qEV columns do not completely eliminate all lipoproteins (e.g., chylomicrons and VLDL), they significantly minimise contamination, particularly from HDL. In contrast, the exoEasy kit exhibits a higher propensity for co-isolation of triglyceride-rich lipoproteins, potentially skewing downstream analyses.

*Note that this workflow was optimised using qEV Legacy columns which have been superseded by qEV Gen 2 columns that can isolate EVs with the lowest lipoprotein contamination.

2. Evaluating Plasma EV Isolation for Prostate Cancer Diagnosis

EVs are emerging as promising biomarkers for liquid biopsy-based cancer diagnosis, but challenges in standardising isolation methods hinder clinical translation. This study compares three commercial kits—two SEC kits (qEV35 and qEV70) and the ExoQuick Ultra polymer precipitation kit—for isolating EVs from human plasma, with a focus on quality, purity, and clinical relevance.

Highlights of the Key Findings

1. Superior Purity with SEC Kits Both qEV35 and qEV70 demonstrated significantly higher EV purity compared to ExoQuick Ultra, which suffered from high levels of lipoprotein and protein contamination. Among the SEC kits, qEV70 provided the highest EV purity, making it ideal for applications requiring minimal contamination.
2. Yield vs. Purity Trade-Off qEV35 offered the highest EV yield but at the expense of lower purity compared to qEV70. Researchers must weigh yield against purity based on downstream applications. For analyses requiring large quantities of EVs, qEV35 may be preferred, while qEV70 is recommended for studies prioritising purity.
3. Challenges with Lipoprotein Contamination Lipoprotein contamination, especially LDL and VLDL, was observed across all methods, with SEC showing better removal of smaller HDL particles.
4. Scalability and Consistency qEV columns enable scalability and reproducibility, which are critical for transitioning EV-based biomarker research into clinical settings.
5. Quantitative Analysis with Particle Numbers qEV70 displayed a linear relationship between particle numbers and EV amount, supporting its use in quantitative EV analysis for clinical diagnostics. This linearity was not observed with ExoQuick Ultra, further highlighting its limitations.

Why qEV Platform Stands Out: Setting the Gold Standard for EV Isolation

The choice of isolation method can make or break the success of EV-based research and clinical diagnostics. The insights gathered from comparative studies underscore why qEV platform remain the benchmark for EV isolation, particularly for applications requiring precision, purity, and scalability.

Key Advantages of qEV Platform:

1. Unmatched Purity: By significantly minimising protein and lipoprotein contamination, qEV platform ensure highly purified EV isolates, crucial for reliable downstream analyses.
2. Tailored Options for Specific Needs: The qEV70 delivers unparalleled purity for sensitive applications, while the qEV35 strikes a balance between yield and quality for projects requiring larger quantities of EVs.
3. Reproducibility and Scalability: Whether in small-scale research or large-scale clinical studies, qEV platform offer consistent and reproducible results, paving the way for seamless translation to clinical settings.
4. Precision in Quantitative Analysis: The linear relationship between particle numbers and EV amount observed with qEV70 makes it a robust tool for quantitative diagnostics, a key requirement for liquid biopsy applications.

Why This Matters

EVs hold immense potential as biomarkers for cancer diagnosis and other diseases. However, the reliability of EV-based research hinges on the quality of the isolated vesicles. qEV platform provides researchers with the confidence that their isolates are as pure and reproducible as possible, addressing key challenges that have historically hindered clinical adoption. By choosing qEV platform, researchers not only enhance the quality of their data but also move a step closer to realising the clinical promise of EV biomarkers.

In a field where precision is paramount, qEV platform stands as a trusted ally for advancing EV research and diagnostics. Ready to elevate your EV isolation process? Learn more about the Izon qEV platform and its applications for liquid biopsy analysis by contacting us directly

References:

Nieuwland R, Enciso-Martinez A, Bracht JWP. Clinical applications and challenges in the field of extracellular vesicles. Med Genet. 2023 Dec 5;35(4):251-258. doi: 10.1515/medgen-2023-2062. PMID: 38835736; PMCID: PMC11006345.

Pang B, Zhu Y, Ni J, Ruan J, Thompson J, Malouf D, Bucci J, Graham P, Li Y. Quality Assessment and Comparison of Plasma-Derived Extracellular Vesicles Separated by Three Commercial Kits for Prostate Cancer Diagnosis. Int J Nanomedicine. 2020 Dec 15;15:10241-10256. doi: 10.2147/IJN.S283106. PMID: 33364756; PMCID: PMC7751609.

Roy, S.; Lin, H.-Y.; Chou, C.-Y.; Huang, C.-H.; Small, J.; Sadik, N.; Ayinon, C.M.; Lansbury, E.; Cruz, L.; Yekula, A.; et al. Navigating the Landscape of Tumor Extracellular Vesicle Heterogeneity. Int. J. Mol. Sci. 2019, 20, 1349. https://doi.org/10.3390/ijms20061349

Simonsen JB. What Are We Looking At? Extracellular Vesicles, Lipoproteins, or Both? Circ Res. 2017 Sep 29;121(8):920-922. doi: 10.1161/CIRCRESAHA.117.311767. PMID: 28963190.

Stranska, R., Gysbrechts, L., Wouters, J. et al. Comparison of membrane affinity-based method with size-exclusion chromatography for isolation of exosome-like vesicles from human plasma. J Transl Med 16, 1 (2018). https://doi.org/10.1186/s12967-017-1374-6