Izon 20 nm Column For Extracellular Vesicle Isolation User Manual

Izon 20 nm Column For Extracellular Vesicle Isolation

Specifications

• Product Name: qEV1 Columns
• Available Sizes: 20 nm, 35 nm Gen 2, 70 nm Gen 2
• Manufacturer: Izon Science Limited
• Extracellular Vesicles Isolation: Rapid and Reliable
• Certification: ISO 13485:2016

Product Usage Instructions

Definitions and Writing Conventions

Refer to the user manual for specific definitions and writing conventions related to the product.

Safety and Hazards

Hazards

Follow all safety guidelines provided in the user manual to avoid potential hazards during product usage.

Storage

Store the qEV1 columns in a cool, dry place away from direct sunlight to maintain product integrity.

Disposal

Dispose of used columns according to local regulations for laboratory waste disposal.

Introduction to Size Exclusion Chromatography

Overview

Size exclusion chromatography is a technique used for separating particles based on their size in a solution.

Intended Use

The qEV1 columns are designed for rapid and reliable isolation of extracellular vesicles.

qEV1 Specifications

Refer to the specifications section above for detailed information on the available sizes and manufacturer details.

qEV1 Performance Characteristics

Consult the user manual for performance characteristics and guidelines for optimal product usage.

Frequently Asked Questions

Can the qEV1 columns be re-used?

A: No, the qEV1 columns are designed for single-use only to ensure accurate and reliable results.

Q: How should I store unused qEV1 columns?

A: Store unused columns in their original packaging in a dry, cool environment away from direct sunlight.

SPECIFICATIONS AND OPERATIONAL GUIDE FOR qEV1 COLUMNS

RAPID & RELIABLE ISOLATION OF EXTRACELLULAR VESICLES

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• Izon Science Limited provides this document to its customers with a product purchase to use in the product operation. This document is copyright protected and any reproduction of the whole or any part of this document is strictly prohibited, except with the written authorisation of Izon Science Limited.
• The contents of this document are subject to change without notice. All technical information in this document is for reference purposes only. System configurations and specifications in this document supersede all previous information received by the purchaser.
• Izon Science Limited makes no representations that this document is complete, accurate or error-free and assumes no responsibility and will not be liable for any errors, omissions, damage or loss that might result from any use of this document, even if the information in the document is followed properly.
• This document is not part of any sales contract between Izon Science Limited and a purchaser. This document shall in no way govern or modify any Terms and Conditions of Sale, which Terms and Conditions of Sale shall govern all conflicting information between the two documents.
• Izon products are designed and manufactured under a quality system certified to ISO 13485:2016.
• All third-party trademarks are the property of their respective owners.

Izon Science Limited PO Box 9292 Addington Christchurch 8024 New Zealand

Telephone: +64 3 357 4270
Email: support@izon.com
Website: www.izon.com

DEFINITIONS AND WRITING CONVENTIONS

Make sure to follow the precautionary statements presented in this guide. Safety and other special notices will appear in boxes and include the symbols detailed below.

Table 1: Safety and Hazard Symbols

Table 2: Terminology Used in this Manual

TERM

| ****

DEFINITION

---|---

Buffer Volume (BV) and Purified Collection Volume (PCV)

| ****

The Buffer Volume (BV) is the discardable eluate prior to the volume containing a high proportion of purified extracellular vesicles (EVs). This EV peak/EV-containing volume is known as the Purified Collection Volume (PCV). Both may differ in volume between resin types and your sample type, so optimisation around our suggested default values is encouraged.

Chromatography

| ****

A method used primarily for separation of the components of a sample. The components are distributed between two phases; one is stationary while the other is mobile. The stationary phase is either a solid, a solid-supported liquid, or a gel/resin. The stationary phase may be packed in a column, spread as a layer or distributed as a film. The mobile phase may be gaseous or liquid.

Column Volume

| ****

The total volume between the upper and lower frits.

Default Buffer Volume

| ****

Relevant when working with the Automatic Fraction Collector (AFC). AFCs have a default buffer volume setting, which can be adjusted to tailor the volume that is collected.

Degassing

| ****

Degassing involves subjecting a solution to vacuum to “boil” off excess dissolved gas e.g. applying a vacuum to a flask.

SAFETY AND HAZARDS

Refer to the Safety Data Sheet for the classification and labelling of hazards and associated hazard and precautionary statements. The Safety Data Sheet for qEV columns is located at support.izon.com/safety-data-sheets

Hazards
qEV columns are a laboratory product. However, if biohazardous samples are present, adhere to current Good Laboratory Practices (cGLPs) and comply with any local guidelines specific to your laboratory and location.

Chemical Hazards

The qEV column contains < 0.1% ProClin 200 or < 0.1% sodium azide*, both of which are harmful if swallowed or in contact with skin.

*Izon is transitioning from the use of sodium azide to ProClin 200 for the storage of qEV columns. For information on how to identify which storage buffer is in your column, visit support.izon.com

Prevention

1. Do not get into eyes, on skin, or on clothing.
2. Wash skin thoroughly after handling.
3. Do not eat, drink, or smoke when using this product.
4. Avoid release of product into the environment.
5. Wear protective gloves and clothing; follow general laboratory precautions.

Response

1. IF SWALLOWED: Immediately call a POISON CONTROL CENTRE/Doctor.
2. IF ON SKIN: Gently wash with plenty of water.
3. Remove immediately any contaminated clothing and wash before reuse.
4. Collect any spillage and dispose of appropriately.

Disposal of Biohazardous Material
Be sure to adhere to the following guidelines and comply with any local guidelines specific to your laboratory and location regarding use and disposal.

General Precautions

• Always wear laboratory gloves, coats, and safety glasses with side shields or goggles.
• Keep your hands away from your mouth, nose, and eyes.
• Completely protect any cut or abrasion before working with potentially infectious or hazardous material.
• Wash your hands thoroughly with soap and water after working with any potentially infectious or hazardous material before leaving the laboratory.
• Remove watches and jewellery before working at the bench.
• The use of contact lenses is not recommended due to complications that may arise during emergency eye-wash procedures.
• Before leaving the laboratory, remove protective clothing.
• Do not use a gloved hand to write, answer the telephone, turn on a light switch, or physically engage with people who are not wearing gloves.
• Change gloves frequently.
• Remove gloves immediately when they are visibly contaminated.
• Do not expose materials that cannot be properly decontaminated to potentially infectious or hazardous material.
• Upon completion of the tasks involving potentially infectious or hazardous materials, decontaminate the work area with an appropriate disinfectant or cleaning solution (1:10 dilution of household bleach is recommended).

Dispose of the following potentially contaminated materials in accordance with laboratory local, regional, and national regulations:

• Biological samples
• Reagents
• Used reaction vessels or other consumables that may be contaminated

Storage
Rapid changes in temperature should be avoided, as this can introduce bubbles into the resin bed.
Store unused qEV columns at room temperature. Used qEV columns can be stored at room temperature providing they have been cleaned according to the instructions in this document and stored using a bactericide or bacteriostatic agent (e.g., PBS containing 0.05% ProClin 200 or 0.05% w/v sodium azide, or 20% ethanol). If the appropriate solutions are not available, store columns at +4 to +8 °C after use.

Disposal
Waste buffer should be disposed of in a safe manner. Sodium azide accumulation over time in copper pipes can result in an explosion.

INTRODUCTION TO SIZE EXCLUSION CHROMATOGRAPHY

Overview
qEV Size Exclusion Chromatography (SEC) columns separate particles based on their size as they pass through a column packed with a porous, polysaccharide resin. As the sample passes through the column under gravity, smaller particles enter the resin pores on their way down and their exit from the column is delayed (Figure 1C). As the sample exits the column, sequential volumes are collected. Particles will be distributed across the volumes based on their size, with the largest particles exiting the column first and the smallest particles exiting the column last. The packed column is equilibrated with a buffer, which fills the column. The total column volume is occupied by both the solid resin (stationary phase) and the liquid buffer (the mobile phase). As the particles do not bind to the resin, the buffer composition will not significantly affect the resolution (the degree of separation between peaks).

Figure 1: Process of Size Exclusion Chromatography (A) Schematic picture of a resin bead with an electron microscopic enlargement. (B) Schematic drawing of sample molecules diffusing into the pores of the resin beads. (C) Graphical description of separation: (i) sample is applied to the column; (ii) the smallest particles (pink) are more delayed than the largest particles (blue); (iii) the largest particles are eluted first from the column. Band broadening causes significant dilution of the particle zones during chromatography. From: GE Healthcare and Biosciences. (n.d.). Size Exclusion Chromatography Principles and Methods [Brochure]. Uppsala, Sweden. Accessed June 2019.

Intended Use
qEV columns are used to isolate extracellular vesicles from biological samples. qEV1 columns are equipped with RFID chips for use with the Automatic Fraction Collector (AFC). These chips will not impact manual use. qEV columns are intended for use by professional personnel only.
qEV columns are designed to isolate and purify vesicles from most biological samples, including:

• Serum
• Plasma
• Saliva
• Urine
• Cerebrospinal fluid
• Cell culture media

NOTE: most ‘raw’ samples cannot be directly run on qEV columns and analysed with tunable resistive pulse sensing (TRPS) without some preparation such as centrifugation and concentration steps. Visit support.izon.com for recommendations and protocols.

qEV1 Specifications
Table 3: qEV1 Specifications

COLUMN SERIES| qEV1/70 nm GEN 2| qEV1/35 nm GEN 2| qEV1/20 nm
---|---|---|---
Default buffer volume| 4.7 mL| 4.7 mL| 4.5 mL
Default PCV| 2.8 mL (4 x 0.7 mL)
Sample load volume| 1 mL
Column volume| 13.5 mL
Optimal fraction size| 0.7 mL
Flush volume| 27 mL
Buffer volume**| 4.0 mL| 4.0 mL| 3.8 mL
PCV| 2.8 mL
Elution peak after buffer volume***| 2.1 mL
Operational temperature| 18 to 24 oC
Buffer| PBS
Upper, lower and loading frit size| 20 µm
pH stability working range| 3–13
pH stability cleaning-in-place (CIP)| 2–14
Shelf-life (if stored correctly)| 12 months

• These are the default values programmed on the Automatic Fraction Collector and have been selected using human plasma samples to offer a balance of EV recovery and high purity. The BV and PCV can be adjusted to suit your sample type and preferences.
• Loading higher sample volumes results in a lower level of purity in the later vesicle volumes, greater overlap between protein and EV elution peaks, and a higher protein peak within the PCV. Loading lower sample volumes results in a higher dilution factor of the sample. The optimal recommended sample volume for purity on the qEV1 is 1 mL.
• Based on Tunable Resistive Pulse Sensing analysis of human plasma samples. N.b., the BV and PCV listed here differ from default recommended values above.

qEV1 Performance Characteristics
When assessing the concentration of particles larger than 60 nm in the PCV of qEV1 columns, the 20 nm column exhibits the highest particle recovery. The 35 nm column ranks second in terms of recovery, and the 70 nm column shows the lowest recovery among them. Among qEV1 columns, EV purity (i.e., the particle- to-protein ratio) is highest in the PCV of 70 nm columns, followed by 35 nm columns. The lowest purity is seen in 20 nm columns.

Different samples may give slightly different elution profiles and purity, hence an initial measurement of EV concentration and protein contaminants in collected fractions is recommended.

Figure 2: Elution profiles of qEV1 columns (20 nm, 35 nm, and 70 nm) with 1 mL of human plasma loaded. Particle concentration was measured using the Exoid, and protein concentration was measured using a bicinchoninic acid (BCA) assay. Particle concentration is expressed as the mean ± standard error, while the percentage of protein recovered is depicted using the mean value. n=3 for 20 nm and 35 nm, n=6 for 70 nm. BV = Buffer Volume; Default PCV = Default Purified Collection Volume.

Choosing a Purified Collection Volume
The optimal PCV will depend on the elution profile of the sample and the applications/analytical methods used downstream. Given the elution profile is impacted by sample characteristics, you should determine the elution profile of your sample to allow you to optimise the PCV. Default values programmed on the AFC (Default BV and Default PCV) are presented in Table 3 and are based on plasma elution profiles (Figure 2).

MANUAL OPERATING INSTRUCTIONS

The following section provides instructions for the manual use of qEV columns. For use of qEV columns with the Automatic Fraction Collector (AFC) instrument, please see the full AFC User Manual at support.izon.com

Operational Recommendations
The following recommendations are provided to ensure optimal performance of the qEV column:
Centrifuge samples prior to loading onto the column. To avoid clogging of column frits, it is recommended to filter or centrifuge the biological sample to remove large particulate matter.

• Centrifuge samples at 1,500 x g for 10 minutes to remove any cells and large particles.

• Gently transfer the supernatant to a new tube and centrifuge again at 10,000 x g for 10 minutes.

• For microvesicle isolation, use lower g-forces for the second centrifugation step.
  Samples can be concentrated before application to the column or after isolation if needed. It is possible to concentrate samples before and/or after use of the qEV column, however Izon offers multiple column sizes to reduce the need for pre-analytical sample concentration. If concentration protocols are needed, please consider the following recommendations:

• Concentration of some sample types may result in the formation of precipitates and protein aggregates, especially for urine samples.

• Concentrated samples should be centrifuged at 10,000 x g for 10 minutes prior to loading onto a qEV column.

• Izon recommends using Amicon® Ultra Centrifugal filters (Merck), and for very large volumes, hollow fibre crossflow filtration. Use according to manufacturer’s recommendations.

Concentration of samples using filtration after purification with qEV may result in the loss of some EVs.

Treating columns as single-use is advisable where the vesicles will be analysed for nucleic acids. This will eliminate the possibility of cross- contamination between samples.
Ensure that the sample buffer has been prepared appropriately. To maintain the functionality of EVs, the flushing buffer should be of the same temperature as the sample buffer. SEC can also be used to exchange the buffer of a sample.

• Sample buffer temperature should be within the operational temperature of 18-24 ˚C (65-75 ˚F).
• Sample buffers should be degassed and room temperature to avoid air bubbles forming in the resin bed.

Rapid changes in temperature, for example removing packed columns from a cold room and applying buffer at room temperature, can introduce air bubbles in the packed bed, resulting in poorer separation.

• Use a buffer with an ionic strength of 0.15 M or greater to avoid any unwanted ionic interactions between the solute molecule and the matrix.
• Only use freshly filtered (0.22 µm) buffer to avoid introducing particulate contamination.
• qEV columns come equilibrated in filtered PBS containing < 0.1% ProClin 200 or < 0.1% w/v sodium azide.

Column Setup and Equilibration

1. Equilibrate the column and the sample buffer to be within the operational temperature range of 18-24 ˚C.
   Do not remove the column caps until the column has reached operational temperature.

2. Carefully remove the top cap only and attach the column in an upright position to a stand ready for use. Alternatively, Automatic Fraction Collectors (AFCs) and qEV racks are available from store.izon. com.

3. Remove the bottom cap and allow the buffer to start running through the column.

Column Flushing

1. Attach the buffer reservoir to the top of the column.
2. Flush the column with at least two column volumes of PBS buffer. This minimises potential effects of storage buffers on your downstream applications. If an elution buffer other than PBS is to be used, equilibrate the column with at least three column volumes of the new buffer.

Only use freshly filtered (0.22 µm) buffer to avoid introducing particulate contamination.

Sample Collection

1. To avoid clogging of column frits, it is recommended to filter or centrifuge the biological sample to remove large particulate matter. See Section 4.1: Operational Recommendations for more information.

2. Continue to allow buffer to run through the column. The column will stop flowing when all of the buffer has entered the loading frit.

3. Load the prepared centrifuged sample volume onto the loading frit.
   Avoid stopping the column flow during the run for long periods of time to ensure accurate EV separation.

4. Immediately start collecting the buffer volume (this includes the volume displaced by loading the sample).

5. Allow the sample to run into the column. The column will stop flowing when all of the sample has entered the loading frit.

6. Top up the column with buffer and continue to collect the buffer volume.

7. Once the buffer volume is collected, continue to collect the Purified Collection Volume (PCV).

To collect accurate volumes, only load the required volume to the top of the column, wait for the volume to run through until the flow stops and repeat.

Column Cleaning and Storage

1. After the desired fractions have been collected, the column should be cleaned and sanitised to remove residual proteins. Rinse the column with 13.5 mL of 0.5 M NaOH directly after finishing fraction collection, then flush with 27 mL of buffer to return the column pH to normal before loading another sample.
   Simply flushing with a large volume of buffer after fraction collection is not sufficient to clean the column completely and there may be some carry-over from previous samples.

2. If storing for future use, store in PBS containing a bactericide or bacteriostatic agent (e.g., 0.05% w/v sodium azide or 0.05% ProClin 200), or 20% ethanol. Columns stored in 20% ethanol should be flushed with two column volumes of DI water after cleaning, then flushed with two column volumes of 20%ethanol for storage. Columns stored in buffer should be flushed with two column volumes of buffer.
   Avoid adding 20% ethanol to buffer inside the column as this can precipitate salt inside the resin bed and damage the column.

3. Columns containing a bacteriostatic agent can be stored at room temperature after use, providing they have been cleaned according to the instructions above. If the appropriate storage solutions are not available then clean columns can be stored at +4 to +8 ˚C after use.

RESOURCES

1. Protocols for EV Isolation from Common Sources
   Visit support.izon.com for application notes and typical protocols for common EV samples. If you are unsure of what to do to prepare your sample, please contact a customer support representative via the contact tab on our website www.izon.com

2. EV Analysis Using TRPS
   Izon recommends TRPS analysis for determination of particle size, concentration, and zeta potential. The TRPS Reagent Kit includes coating solutions for pre-coating the pore, minimising non-specific binding and provides for stable and accurate size and concentration analysis.
   For TRPS analysis of the EVs, Izon recommends an initial dilution of 1/5 or 1/10 in electrolyte. Optimise the dilution to achieve a rate at the middle operating pressure of approximately 500 to 1500 particles per minute to avoid pore blockage.
   Visit support.izon.com for more information on the analysis of EVs with TRPS.

3. Kits for EV Concentration and qEV RNA Extraction
   To support broader analyses downstream of qEV isolation, Izon offers the qEV Magnetic Concentration Kit and qEV RNA Extraction Kit. The qEV Magnetic Concentration Kit uses functionalised particles which bind irreversibly to EVs, and can be used to concentrate EVs for protein analysis (mass spectrometry or Western blot) or RNA analysis (PCR or RNA sequencing). The qEV RNA Extraction Kit can be used separately, or together with the qEV Magnetic Concentration Kit. To learn more visit izon.com/qev/kits

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References

• Introducing the qEV Concentration kit and the qEV RNA Extraction Kit
• Izon Science Support Centre
• Isolate & Measure Extracellular Vesicles & Nanoparticles

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