IZON ICS-70 qEV Single Size Exclusion Chromatography Columns User Manual

: June 4, 2024
: IZON

IZON ICS-70 qEV Single Size Exclusion Chromatography Columns

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Izon Science Ltd
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 in Table 1.

Table 1: Safety and Hazard Symbols

| This symbol indicates general advice on how to improve procedures or recommends measures to take in specific situations.
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| This symbol indicates where special care should be taken.

Table 2: Terminology Used in this Manual

TERM	DEFINITION
Buffer volume (Previously void volume)	**** The volume of liquid that

corresponds to the volume before the Purified Collection Volume (PCV); this volume is collected automatically into the central well. The buffer volume is different for each column type.
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 one is mobile. The stationary phase is either a solid, a solid-supported liquid, or a gel. 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 combined volume of packed material and buffer volume (can be referred to as the bed volume).
Degassing| Degassing involves subjecting a solution to vacuum to “boil” off excess dissolved gas e.g. applying a vacuum to a flask.
Flow rate| The volumetric flow in mL/min of the carrier liquid.
Purified Collection Volume (PCV)| **** The volume immediately succeeding the buffer volume, containing particles of interest purified from the loaded sample. The PCV is different for each column type.

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 Isolation columns is located at www.izon.com/resources

Hazards

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

Disposal of Biohazardous Material
The qEV column contains < 0.1% sodium azide, which is potentially fatal if swallowed or in contact with skin. Please review the following guidelines and precautions prior to each use of the qEV column:

Prevention:

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

Response

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

For more information, see the MSDS Documentation for Izon qEV columns: www.izon.com/resources

| Sodium azide can be fatal if swallowed or in contact with skin. It can cause damage to neurological organs through prolonged or repeated exposure. It is very toxic to aquatic life with long-lasting effects.
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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 without 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 gel bed.

Unused qEV columns can be stored at room temperature.

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 SEC (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 (yellow) are more delayed than the largest particles (red); (iii) the largest particles are eluted first from the column. Band broadening causes significant dilution of the particle zones during chromatography. (D) Schematic chromatogram. From: GE Healthcare and Biosciences. (n.d.). Size Exclusion Chromatography Principles and Methods [Brochure]. Uppsala, Sweden. Accessed June 2019.

Intended Use

Izon qEV columns isolate extracellular vesicles from biological samples.
qEVsingle columns are equipped with RFID chips for use with the Izon Automatic Fraction Collector (AFC). These chips will not impact manual use. The column is intended to be used in research laboratories by professional personnel for research use only. The qEV column is not intended for diagnostic purposes and should not be used to make treatment decisions.

qEV columns are designed to isolate and purify vesicles from most biological samples, including:

Serum
Plasma
Saliva
Urine
Cerebrospinal Fluid (CSF)
Cell culture media

NOTE: most ‘raw’ samples cannot be directly run on qEV columns and analysed with TRPS without some preparation such as centrifugation and concentration steps. Contact the Izon Support Centre for recommendations and protocols.

Comparison of qEV/35nm and qEV/70nm series

All qEV columns are available in one of two isolation ranges, the qEV/35nm series and the qEV/70nm series. The qEV/35nm series of columns generally perform better when the target particle to be isolated is less than 110 nm in diameter, while the qEV/70nm series of columns generally perform better when the target particle to be isolated is greater than 110 nm in diameter ( see Table 3 ). For optimal recovery of particles between 35 and 350 nm a qEV/35nm series column is recommended. For optimal recovery of particles between 70 and 1000 nm a qEV/70nm series column is recommended.

Figure 2. Particle recovery by size for qEV/35 series column compared to qEV/70 series column (pink line).

Table 3: Specifications of qEV/35nm and qEV/70nm Series

	qEV/35nm SERIES	qEV/70nm SERIES
**** Target Particle Size	35 to 350 nm	**** 70 to 1000 nm
Optimum Recovery Range	Particles < 110 nm	**** Particles > 110 nm

qEVsingle Specifications

Table 4: qEVsingle Specifications

Column name|

qEVSINGLE

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Column series|

qEVSINGLE /35nm

|

qEVSINGLE /70nm

Optimal separation size|

35 – 350 nm

|

70 – 1000 nm

Sample load volume|

150 µL*

Column volume|

3.5 mL

Optimal fraction size|

0.2 mL

Buffer volume***|

0.8 mL

|

1 mL**

Flush volume|

7 mL

Purified Collection Volume***|

0.6 – 0.8 mL

EV peak after buffer volume***|

0.3 ± 0.1 mL

|

0.5 ± 0.1 mL

Operational temperature|

18 to 24 °C

Buffer|

PBS

Largest size passable|

1 µm

Top and bottom frit size|

20 µm

pH stability working range|

3 – 13

pH stability cleaning-in-place (CIP)|

2 – 14

Shelf life|

12 months

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 qEVsingle is 150 µL.

**EVs and similarly sized particles have been detected from 0.8 mL on a qEVsingle/70 column, however the concentration is extremely low and may cause significant sample dilution if included.

***Values for human plasma samples only.

qEVsingle Performance Characteristics

As shown in Figure 3 and 4 below, particles less than 70 nm typically elute later than the PCV on the qEV/70nm, whereas particles larger than 35 nm are captured in this zone on the qEV/35nm column. A higher recovery in the PCV of particles larger than 70 nm occurs on the qEV/70nm series columns compared with the qEV/35nm series (Fig. 3). Proteins and EVs typically elute slightly earlier on the qEV/35nm series, hence a buffer volume of 0.8 mL. Higher protein levels in the PCV are mainly due to an increase in EV bound proteins.

| 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.
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Figure 3: Comparison of total protein elution levels and recovery of 69 nm liposomes between a qEVsingle/35nm and a qEVsingle/70nm.

Figure 4: Comparison of total protein levels and concentration of extracellular vesicles (EVs) and similarly sized particles >60 nm on a qEVsingle/70nm versus a qEVsingle/35nm. Particles measured using TRPS, protein measured using BCA assay.

qEVsingle EV Elution Profile

The elution of vesicles typically peaks at 600 µL ± 200 µL after the buffer volume for a 150 µL sample volume and collecting 200 µL fractions. Figure 5 shows the elution of vesicles when 150 µL of human plasma sample is loaded onto a qEVsingle/70nm column.

The majority of EVs typically elute in 1000 µL after the buffer volume. If higher purity is desired, collect only the first 600 µL. The user therefore chooses between maximising recovery by collecting a bigger volume or maximising purity by collecting a lesser volume.

The elution of plasma protein is slower, eluting predominantly from 0.8 to 4 mL after the buffer volume. Any vesicles recovered beyond 0.8 mL contains higher protein contamination and may be less suitable for downstream analysis because of their lower purity.

Figure 5: Typical elution profile for a qEVsingle column with 150 µL of plasma loaded; proteins elute later than extracellular vesicles (EVs) and similarly sized particles >60 nm.
Particles measured using TRPS, protein measured using BCA assay.

qEVsingle Sample Input Volume Effects and Recovery Rates

Effect of Resin Type on Protein Elution Profile

The PCV on a qEVsingle contains very little protein, with protein levels increasing in later volumes, as shown in Figure 6. Higher protein levels for the qEVsingle/35nm are due in part to higher recovery of smaller EVs in the PCV (refer to Fig. 2).

Figure 6: Percentages of loaded protein in fractions after the buffer volume, loading 0.15 mL of human plasma onto a qEVsingle. Pink bars are the optimised PCV for a human plasma sample and covers a total of 0.8 mL. qEVsingle/70nm bars are dark pink and dark blue. qEVsingle/35nm bars are light pink and light blue.

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 move the supernatant to a new tube and centrifuge again at 10,000 x g for 10 min.
- 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 both 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 Merck Millipore concentration devices (Amicon® Ultra Centrifugal filters; C7715). Use according to manufacturer’s recommendations.
- Concentration of samples after purification with qEV may result in the loss of some EVs on the membrane.
Single-use columns are advisable where the vesicles will be analysed for nucleic acids. Use of a single-use column, such as the Izon qEVsingle, will reduce the possibility of cross-contamination.
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 gel 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% w/v sodium azide.

Column Setup and Equilibration

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.
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| Sample buffers should be degassed and room temperature to avoid air bubbles forming in the gel 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.
Carefully remove the top cap only and attach the column in an upright position to a stand ready for use. qEVsingle racks and the Automatic Fraction Collector (AFC) are available from store.izon.com
Remove the bottom cap and allow the buffer to start running through the column.
If you are using PBS as your buffer then your column is ready to use. If your downstream application is affected by 0.05% sodium azide, flush the column with at least two column volumes of PBS.
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
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Sample Loading

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.
Continue to allow buffer to run through the column. The column will stop flowing when all of the buffer has entered the loading frit.
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.
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Immediately start collecting the buffer volume (this includes the volume displaced by loading the sample).
Allow the sample to run into the column. The column will stop flowing when all of the sample has entered the loading frit.
Top up the column with buffer and continue to collect the buffer volume.
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.
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Column Disposal

qEVsingle columns are specifically designed for single use and should be safely disposed after use.

RESOURCES

Column Cleaning and Sanitisation

qEVsingle columns are specifically designed for single use.

Protocols for EV Isolation from Common Sources

See Izon Support Centre 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 support@izon.com for assistance.

EV Analysis Using TRPS

Izon recommends TRPS analysis for determination of particle size, concentration, and zeta potential. The Izon Reagent Kit includes coating solutions for pre-coating the pore, minimising non-specific binding and provides for stable and accurate sizing 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 highest operating pressure of approximately 200 to 1600 particles per minute to avoid pore blockage.

See Izon Support Centre support.izon.com for more information on the analysis of EVs with TRPS.