Agilent AdvanceBio SEC 1.9 Column User Guide PDF

Agilent AdvanceBio SEC 1.9 m Columns User guide

Agilent size exclusion columns optimized for separation and

characterization of size variants are offered in two pore sizes: AdvanceBio

SEC 120 is best suited for analysis of peptides and small therapeutic

proteins; AdvanceBio SEC 200 was designed for characterizing mAbs and

ADCs. In addition to 4.6 mm id stainless steel columns, AdvanceBio SEC

1.9 m is also available in PEEK-lined stainless steel columns in 2.1 mm id.

These bio-inert columns provide a metal-free flow path and are particularly

suited to SEC-MS applications.

www.agilent.com/chem/advancebio

DE44376.2784953704

This information is subject to change without notice.

Agilent Technologies, Inc. 2021 Published in the USA, July 9, 2021 5994-0739EN

Ordering details

Description Part Number

AdvanceBio SEC 120 1.9 m, 4.6 300 mm PL1580-5250

AdvanceBio SEC 120 1.9 m, 4.6 150 mm PL1580-3250

AdvanceBio SEC 120 1.9 m guard, 4.6 30 mm PL1580-1250

AdvanceBio SEC 200 1.9 m, 4.6 300 mm PL1580-5201

AdvanceBio SEC 200 1.9 m, 4.6 150 mm PL1580-3201

AdvanceBio SEC 200 1.9 m guard, 4.6 30 mm PL1580-1201

AdvanceBio SEC 120 1.9 m, 2.1 150 mm, PEEK PL1980-3250PK

AdvanceBio SEC 120 1.9 m, 2.1 50 mm, PEEK PL1980-1250PK

AdvanceBio SEC 200 1.9 m, 2.1 150 mm, PEEK PL1980-3201PK

AdvanceBio SEC 200 1.9 m, 2.1 50 mm, PEEK PL1980-1201PK

Please see www.agilent.com for PEG, PEO, and polysaccharide mol wt calibration standards.

Recommended storage

Short-term storage (less than two weeks): store the column in the mobile phase.

Extended storage (longer than two weeks): store the column in filtered 100 mM sodium phosphate, pH 7, with or without 0.02% NaN3, or 20% methanol in water. Flush the column with a minimum of 10 column volumes. To switch to or from 20% methanol, column flushing must be done at low flow rates to avoid overpressuring the column due to high viscosity. Starting at a lower flow rate, flush at no more than 0.1 mL/min for 4.6 mm columns, and no more than 0.05 mL/min for 2.1 mm columns, being sure to keep the pressure below 400 bar.

Store columns at room temperature.

Column care and cleaning

Column care

An increase in backpressure and a decrease in performance may occur over time. If the pressure has increased, first identify if this increase is due to a guard column that may need to be replaced. If the increase in pressure is in a system component, such as tubing or a filter, replace the component and retest.

Column cleaning instructions

It may be possible to restore column performance using one of the following cleaning solutions:

For strongly adsorbed contaminants: high salt concentration at low pH (for example, 0.5 M Na2SO4, pH 3) or 0.5 M guanidine hydrochloride

Organic solvent for hydrophobic materials: up to 50% methanol, ethanol, or isopropanol

Acidic reagents for basic contaminants: 0.1% TFA, formic acid, or acetic acid in 15% acetonitrile

Always flush the column in the direction of the flow arrow, and adjust the flow rate to keep the pressure below 400 bar. Rinse with at least five column volumes of ultrapure water before and after flushing with at least 20 column volumes of the cleaning solution.

It is not recommended to use all three cleaning buffers sequentially. Choose the most appropriate buffer for your probable contaminant. Take care to avoid precipitation of buffer salts, and avoid overpressuring the column due to mobile phase viscosity differences.

Parameter Value

Column AdvanceBio SEC 200 , 4.6 300 mm, 1.9 m (p/n PL1580-5201)

Instrument Agilent 1260 Infinity II Bio-inert LC system

Flow Rate 0.35 mL/min

Mobile Phase 150 mM sodium phosphate, pH 7.0

Wavelength 280 nm

Column Temperature

25 C

Sample A stressed mAb (1 g, injected onto column) mAb sample stressed in 100 mM sodium bicarbonate pH 9 and incubated overnight at 40 C

0

0

Acquisition time (min)

mAU

5

10

15

20

25

2

5 6 7 8

4 6 8 10 12

Aggregates

Monomer

Fragments

Recommended starting conditions

Important safety considerations All connection points in an LC system are potential

sources of leaks. Users should be aware of the potential toxicity or flammability of their mobile phases.

Do not remove the column end fittings.

Using your column

Installation

Remove both end plugs, and ensure that your systems flow direction matches the arrow on the column. Do not use the column with the flow in the reverse direction.

Use an Agilent InfinityLab Quick Connect LC fitting (p/n 5067-5966) to quickly connect the column to your LC. For PEEK-lined 2.1 mm id columns, Bio-inert UHP-FF fittings (p/n 5067-5695) along with a tool to avoid over-tightening (p/n 5043-0915) are recommended.

Column conditioning

The columns are shipped in 100 mM sodium phosphate buffer, pH 6.7, containing 0.02% NaN3, and must first be flushed into the mobile phase required for your separation. Ramp up the flow rate slowly from 0.0 mL/min to the intended operating flow rate over a period of several minutes. If possible, the maximum flow gradient should be set at 0.1 mL/min/min. Equilibrate the column by flushing for a minimum of 10 column volumes or until the baseline is stable.

Getting started A column performance report, including a column-specific quality control (QC) test chromatogram and a batch- specific protein separation, is enclosed with every Agilent AdvanceBio SEC 200 1.9 m column. The QC test system has been modified from a standard system to minimize dead volume, so it may vary from the system used in your lab. This modification enables better evaluation of the column efficiency, and ensures a more consistent product. An optimized LC system will generate similar results to the chromatogram in the column performance report. For the best chromatographic results, it is recommended that a low dispersion LC is used. You can optimize your LC for maximum resolution by minimizing tubing internal diameter (id) and length between the sample injector and the column and between the column and detector. Low volume micro UV detector flow cells may be necessary with 2.1 mm id columns.

Ensuring proper column connection is important. Agilent recommends InfinityLab Quick Connect LC fittings (p/n 5067-5966), or Bio-inert UHP-FF fittings (p/n 5067-5695), particularly for use with PEEK-lined 2.1 mm id columns to avoid damage.

To monitor column and instrument performance, Agilent recommends running a standard test mixture regularly, such as the AdvanceBio SEC standards.

Column specifications

Parameter Value

Shipping Solvent/Long-Term Storage Solution

pH 6.7 100 mM sodium phosphate buffer with 0.02 % NaN3

Working Flow Rate for 4.6 150 mm 0.1 to 0.7 mL/min for 4.6 300 mm 0.1 to 0.5 mL/min for 2.1 mm id columns 0.05 to 0.10 mL/min

Injection Volume 15 L (recommended) Maximum 1% column volume

Maximum Pressure 620 bar (9,000 psi)

pH Stability 2 to 8.5

Salt Concentration 0.5 M

Mobile Phase Compatibility Compatible with all the SEC mobile phases for UV. Phosphate buffer, pH 7.0 with different salt concentrations and denaturing and native mode SEC-MS mobile phases.

Operating Temperature 20 to 40 C (recommended), 80 C (maximum)

Note: Working at extremes of the operating parameters may reduce column lifetime.0

0

0

1

1

2

2

3

3

4

4

5

5

Norm.

200

400

600

800

2 4 6 8 10 12

1. 2. 3. 4. 5.

Thyroglobulin 670,000 -Globulin 158,000 Ovalbumin 44,000 Myoglobin 17,000 Angiotensin II 1,000

Agilent SEC 300 Standard

Molecular Weight

1. 2. 3. 4. 5.

Ovalbumin 44,000 Myoglobin 17,000 Aprotinin 6,700 Neurotensin 1,700 Uridine 244

Low mol wt Protein Mix

Molecular Weight

2 4 6 8 10 12 min

min

mAU

0

200

400

600

800

1,000

AdvanceBio SEC 200

AdvanceBio SEC 120

Parameter Value

Columns AdvanceBio SEC 200, 4.6 300 mm, 1.9 m (PL1580-5201) AdvanceBio SEC 120, 4.6 300 mm, 1.9 m (PL1580-5250)

Flow Rate 0.35 mL/min

Mobile Phase 150 mM sodium phosphate, pH 7.0

Wavelength 220 nm

Column Temperature

Ambient

Instructions for use

Columns are compatible with commonly used aqueous buffers, including 150 mM sodium phosphate at pH 7.0 with or without the addition of other salts. Salt concentration should not be more than 0.5 M. It is recommended that the percentage of organic solvent be less than 50%. For native mode SEC-MS, ammonium acetate is recommended. Flush the column extensively before connecting to your MS detector in case nonvolatile mobile phase salts are still present. When changing eluents, always consider the viscosity and risk of salt precipitation. If you are unsure, flush the column first with high-purity water before introducing a new eluent.

Mix your buffers freshly using high-purity components and ultrahigh purity water, such as Milli-Q or Nanopure. Filter buffers through a 0.2 or 0.45 m filter, and degas before use. Filtering will remove particulates, and help reduce the risk of bacterial growth, which will otherwise damage the column and your LC system.

Prepare your samples in the mobile phase, and ensure that they dissolve completely. Filter or centrifuge samples before injection.

Note: To maximize the lifetime of your column, we recommend using an Agilent AdvanceBio SEC guard column.

The AdvanceBio SEC 200 1.9 m columns are recommended for use with UV, DAD, LS detectors, and for SEC-MS applications under denaturing and native mode.

If you have specific questions, contact Agilent technical support a