BIO BUNDLE - Phenomenexphx.phenomenex.com/lib/BR15050713_W.pdf · various HPLC column vendors....

BIO BUNDLEChromatography Solutions for BioSeparationsCost-Effective UHPLC/HPLC/GFC Solutions for • mAbs • Proteins • Peptides • Oligonucleotides

Phenomenex l WEB: www.phenomenex.com

Featuring:

• Ultra-High Performance Chromatography Solutions

• Reversed Phase Method Development Strategies for Proteins and Peptides

• Better Understanding of Gel Filtration Chromatography (GFC) Principles

Reversed Phase UHPLC/HPLC

Size Exclusion/Gel Filtration

p. 15 ....Developing Protein Aggregation Methods (TN-1131)

p. 20 ....Better Gel Filtration: Method Development Parameters (TN-1156)

Core-Shell Technology Designed to Bring Ultra-High Resolution and Peak Capacity to Bioseparations

Ultra-High Resolution at an Unbelievable Price

p. 5 ......Improved Intact IgG Separations (TN-1110)

p. 8 ......Improving Biogeneric Protein Separations (TN-1111) Comparison of Aeris WIDEPORE Versus ZORBAX® 300SB

p. 12 ....Improved Peptide Mapping (TN-1124)

Trademarks ZORBAX is a registered trademark of Agilent Technologies, Inc.

Disclaimer Phenomenex is in no way affiliated with Agilent Technologies, Inc. Comparative separations may not be representative of all applications.

© 2013 Phenomenex, Inc. All rights reserved.

Additional Resources

• www.phenomenex.com/Aeris

• Quick Overview

• Product Guide

• Method Optimization Tips

• Column Care & Use

• www.phenomenex.com/Yarra

• Product Guide

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5For additional technical notes, visit www.phenomenex.com

TN-1110

Achieving Improved Intact IgG Separations Using Aeris™ WIDEPORE Core-Shell HPLC | UHPLC ColumnsMichael McGinley, Deborah Jarrett, and Jeff Layne Phenomenex, Inc., 411 Madrid Avenue, Torrance, CA 90501 USA

Aeris WIDEPORE core-shell HPLC | UHPLC columns provide dramatic improvement in the separation of intact antibodies when compared to fully porous columns. The high permeability of the Aeris core-shell particle combined with low hydrophobicity and inert surface results in high recoveries and improved peak shape for even the most hydrophobic proteins.

IntroductionThe use of core-shell technology has resulted in a new paradigm in ultra-high performance by decoupling increased column effi-ciency from high backpressure. The principal result for most small molecule applications has been reduced run times and increased throughput without the need for expensive new UHPLC instrumen-tation.

However, for intact protein separation, the principal focus is more on improving resolution of proteins from near-identical post-trans-lationally modified impurities rather than reducing run times. A wide pore core-shell HPLC | UHPLC column (Aeris WIDEPORE) has been introduced that is specifically designed to improve protein separations. Rather than utilize a similar morphology of small mol-ecule core-shell columns with larger pores, a completely different particle was developed that takes into account the slower diffusion of proteins into porous particles. A graphic representation of the Aeris particle is shown in Figure 1.

When one looks at various intact protein separations, the resolution of different glycoforms of therapeutic IgG antibodies stands out as one of the more difficult due to the large size and structure of IgG. In this technical note, improved separation of IgG glycoforms are shown using Aeris core-shell HPLC | UHPLC columns.

Materials and MethodsAll chemicals, standards and antibodies were obtained from Sig-ma Chemical (St. Louis, MO. USA). Solvents were purchased from EMD (San Diego, CA, USA). Fully porous 5 µm 300 Å C18 columns and core-shell Aeris 3.6 µm WIDEPORE XB-C18 columns (100x4.6 mm) were obtained from Phenomenex (Torrance, CA. USA).

Mouse Immunoglobulin IgG samples were analyzed on an Agilent® 1200 HPLC system with autosampler, column oven, solvent de-gasser, and UV detector set at 214 nm. Data was collected us-ing Chemstation software (Agilent, Santa Clara, CA. USA). Mobile phases used were 0.1 % TFA in water (A) and 0.1 % TFA in aceto-nitrile and a gradient from 10 to 40 % B in 15 minutes was used at 1 mL/min. Column was maintained at 80 °C.

Results and DiscussionAs is shown in Figure 1, Aeris WIDEPORE 3.6 µm is a significantly different particle morphology compared to small pore core-shell media (Kinetex® 2.6 µm, for example, uses a 0.35 µm shell on a 1.9 µm core). Aeris WIDEPORE core-shell particles were designed to maximize resolution of proteins greater than 10 kilodaltons mo-lecular weight regardless of whether an HPLC or UHPLC is used. The thin porous shell minimizes protein peak band spreading due to diffusion in and out of the porous layer; the larger particle size re-duces column backpressure allowing for the use of longer columns for increased resolution. The result is a column with performance on par or better than sub 2 µm wide pore fully porous media at backpressures significantly lower than 3 µm fully porous columns.

The performance advantage of Aeris WIDEPORE core-shell col-umns is demonstrated in Figure 2 where an Aeris column is com-pared to a 5 µm fully porous wide pore column. IgG immunoglobu-lins are considered difficult proteins to separate by reversed phase HPLC due their large size (150 KDa) and hydrophobicity. Typical-ly, elevated column temperatures and isopropanol mobile phase are required to improve recovery and resolution. In this example, mouse immunoglobulin IgG separation is compared on each col-umn using an acetonitrile-only mobile phase at 80 °C. (Aeris col-umns are stable to 90 °C) Note the significantly narrower peak width for the Aeris WIDEPORE core-shell column resulting in the resolution of the three main glycoforms of IgG versus the fully po-rous columns where only two components are baseline resolved. Of additional note is the greater recovery for the Aeris column; low hydrophobicity and good inertness results in greater recovery for hydrophobic proteins.

3.6 µm Core-Shell Particle

Figure 1. Graphical representation of an Aeris WIDEPORE 3.6 µm particle. A 0.2 µm porous shell surrounds a 3.2 µm solid core. This particle geometry is specifically designed to narrow the peak width and improve resolution for proteins and other large molecules.

Figure 2. Comparison between a fully porous 5 µm 300 Å C18 column (top chro-matogram) to an Aeris WIDEPORE 3.6 µm XB-C18 column (bottom chro-matogram) for a mouse IgG sample. Note the significantly narrower peak width and greater protein recovery for the Aeris column. The thin shell minimizes protein diffusion distance resulting in narrower peak widths and greater resolution of closely related species.

min0 2 4 6 8 10 12 14

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19841

App

ID 1

9841

Aeris WIDEPORE 3.6 µm XB-C18 core-shell column

min0 2

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02.5

57.510

12.515

17.520

App

ID 1

9852

Fully Porous 5 µm 300 Å C18 column

3.6---3.2 solid core 3.6---2.6 solid core 1.7---1.25 solid core

0.2 µm Porous Shell

3.6 µm

3.2 µm Solid Core

™

6

If Aeris core-shell technology does not provide at least an equiva-lent separation as compared to other products of the same phase and dimensions, return the product with comparative data within 45 days for a FULL REFUND.

Terms and Conditions Subject to Phenomenex Standard Terms and Conditions, which may be viewed at www.phenomenex.com/TermsAndConditions.

Trademarks Aeris is a trademark, and Kinetex is a registered trademark of Phenomenex, Inc. Agilent is a registered trademark of Agilent Technologies, Inc.


© 2013 Phenomenex, Inc. All rights reserved. TN90

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ConclusionThe good resolution and recovery for this application demonstrate the utility of using core-shell Aeris™ WIDEPORE columns for immu-noglobulin and large protein separations.

Aeris WIDEPORE 3.6 µm Minibore Columns (mm)50 x 2.1 100 x 2.1 150 x 2.1 250 x 2.1

XB-C18 00B-4482-AN 00D-4482-AN 00F-4482-AN 00G-4482-ANXB-C8 00B-4481-AN 00D-4481-AN 00F-4481-AN 00G-4481-ANC4 00B-4486-AN 00D-4486-AN 00F-4486-AN 00G-4486-AN

Aeris WIDEPORE 3.6 µm Analytical Columns (mm)100 x 4.6 150 x 4.6 250 x 4.6

XB-C18 00D-4482-E0 00F-4482-E0 00G-4482-E0XB-C8 00D-4481-E0 00F-4481-E0 00G-4481-E0C4 00D-4486-E0 00F-4486-E0 00G-4486-E0

For more information on Aeris Core-Shell HPLC | UHPLC columns visit www.phenomenex.com/Aeris

Ordering Information

TN-1157

Gain immediate performance advantages on your current method by switching to Aeris. It’s THAT simple. www.phenomenex.com/AerisPhenomenex products are available worldwide. Email us at [email protected].

Protein and Peptide

Separation

POWER

™

TM

Introducing Aeris™ core-shell HPLC | UHPLC columns for proteins and peptides. A new family of core-shell particles specifically designed to bring ultra-high resolution and peak capacity to bioseparations on ANY system.

http://www.phenomenex.com/Aeris

8

TN-1111

Improving Intact Biogeneric Protein Separations with Aeris™ WIDEPORE Core-Shell ColumnsMichael McGinley, Deborah Jarrett, and Jeff Layne Phenomenex, Inc., 411 Madrid Avenue, Torrance, CA 90501 USA

By greatly reducing the path length of protein diffusion, protein peaks tend to be narrower allowing for better resolution between intact proteins and their post-translationally modified impurities. The larger particle size of Aeris WIDEPORE columns generates lower column backpressures than small particle fully porous wide pore columns. This lower column backpressure enables the use of longer columns for maximizing protein resolution.

In recent years several therapeutic proteins have gone off patent, allowing a large number of organizations to develop their own ge-neric versions of these potent therapeutics. With bioequivalence being an important aspect in the regulatory success of any biother-apeutic it is of the utmost importance for researchers to develop analytical methods that fully characterize and quantitate all of the impurities present in a candidate molecule. While peptide mapping is the more common method for identifying low-level post-trans-lational modifications (PTMs), mapping gives only minimal infor-mation about protein folding and can sometimes miss N-terminal and C-terminal modifications. Thus, intact protein analysis by re-versed phase HPLC | UHPLC is usually part of the suite of testing performed on any protein therapeutic. The Aeris WIDEPORE core-shell column offers a new and improved solution for intact protein analysis by offering narrower peak widths and improved protein resolution when compared to fully porous wide pore media. This technical note will show several examples of using Aeris WIDE-PORE for analyzing PTM’s on small to moderate proteins similar in size and chemical characteristics to several protein therapeutics. In addition, separations on common biogenerics will also be shown to demonstrate the improved performance of the Aeris WIDEPORE core-shell columns.

Materials and MethodsAll chemical and standard proteins were obtained from Sigma Chemical (St. Louis, MO, USA). Recombinant human EGF and alpha interferon were purchased from R&D Systems (Minneapolis, MN, USA). Solvents were purchased from EMD (San Diego, CA, USA). Fully porous 300 Å C18 columns were purchased from various HPLC column vendors. Core-shell Aeris WIDEPORE 3.6 µm XB-C18 columns were obtained from Phenomenex (Torrance, CA, USA).

Myoglobin samples were partially degraded by incubation at room temperature for up to a week in dilute acid. Ribonuclease samples were reduced with 100 mM DTT in 50 mM NH4HCO3 pH 8.0 for 20 minutes at 45 ºC; reduced/non-reduced mixtures were generated by spiking different ratios of the native to the reduced sample pri-or to injection on HPLC. Different protein samples were analyzed on an Agilent® 1200 HPLC system with autosampler, column oven, solvent degasser, and UV detector set at 214 nm. Data was collect-ed using ChemStation software (Agilent, Santa Clara, CA, USA). Mobile phases used were 0.1% TFA in water (A) and 0.085% TFA in acetonitrile (B). Different gradients, flow rates and column tempera-tures were listed with the corresponding chromatograms.

Aeris WIDEPORE is a recently introduced core-shell HPLC | UHPLC column specifically designed to provide improved resolution of intact proteins larger than 10 kilodaltons (KDa) in molecular weight. The improved resolution of proteins is accomplished by the use of a new core-shell particle morphology which minimizes protein band-spreading that occurs during diffusion in and out of the core-shell particle. The result is narrower peaks and better resolution of closely eluting proteins. This improved resolution is especially useful for refolding, impurity, and post-translational modification assays on intact biogeneric proteins where very slight differences between intact and modified proteins elute closely on reversed phase columns. Several examples are shown demonstrating the utility of Aeris WIDEPORE core-shell columns for such applications.

IntroductionSince their debut over two years ago, Kinetex® core-shell columns have introduced a new paradigm in ultra-high performance by decoupling column efficiency from high backpressures. This has resulted in small molecule applications with reduced run times and increased throughput without the need for expensive new UHPLC instrumentation.

While analysis speed has some value for protein separations, the principal focus is more on improving resolution of proteins from near-identical post-translationally modified impurities rather than reducing run times. A new wide pore core-shell column specifically designed to improve protein separations (Aeris WIDEPORE) has been introduced. Rather than utilize a similar morphology of small molecule core-shell columns with larger pores, a completely different particle that takes into account the slower diffusion of proteins in and out of porous particles was developed. A graphic representation of the Aeris particle is shown in Figure 1.

3.6 µm Core-Shell Particle

3.6---3.2 solid core 3.6---2.6 solid core 1.7---1.25 solid core

0.2 µm Porous Shell

3.6 µm

3.2 µm Solid Core

™

Figure 1. Graphical representation of Aeris 3.6 µm WIDEPORE particle. A 0.2 µm porous shell surrounds a 3.2 µm solid core. This particle geometry is specifically designed to narrow the peak width and improve resolution of proteins and other large molecules.

TN-1111

9

TN-1111

Conditions for both columns:Column: Aeris WIDEPORE 3.6 µm XB-C18

ZORBAX® 300SB 3.5 µm C18Dimensions: 150 x 4.6 mm

Mobile Phase: A: Water with 0.1 % TFAB: Acetonitrile with 0.1 % TFA

Gradient: A/B (97:3) for 3 min to A/B (35:65) over 30 minFlow Rate: 1.5 mL/min

Temperature: 40 ºCInjection Volume: 20 µL

Instrument: Agilent® 1200SLDetection: UV @ 210 nm (ambient)

Sample: Degraded Myoglobin

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AppID 19837

App

ID 1

9837

150 bar

*Agilent® ZORBAX® 300SB 3.5 µm C18

Figure 2. Comparison between the Aeris WIDEPORE 3.6 µm XB-C18 and a 300 Å fully porous 3.5 µm C18 column. A degraded myoglobin sample was used to compare performance between the two columns. Note the increase in resolution and narrow peak width of the multiple impurities partially resolved on the Aeris core-shell column. In both cases a 150 x 4.6 mm column was used. Flow rate was 1.5 mL/min and column temperature was 40 °C. Gradient was from 3 to 65 % B in 30 minutes after a 3 minute hold.

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AppID 19837

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9836

Aeris WIDEPORE 3.6 µm XB-C18

150 bar

Dramatically Improved Resolving Power

Results and DiscussionIntact protein analysis is performed on recombinant proteins to quantitate the purity of a protein and potentially identify any spe-cific impurities in a sample. For most purified proteins the typical impurity is a post-translationally modified version of the protein or an improperly folded species of the protein. Since such PTM impu-rities are chemically similar to the intact therapeutic protein, achiev-ing chromatographic separation by HPLC or UHPLC between the two species can be a challenge. Separation and quantitation of PTM proteins can be especially difficult for larger proteins since chemical differences induced by a single modification have a small-er net effect. For effective quantitation of impurities, a wide pore re-versed phase media must maximize both efficiency and selectivity.

Aeris™ WIDEPORE, a recently introduced line of core-shell HPLC | UHPLC columns, utilizes a unique particle morphology specifically designed to reduce peak broadening resulting from slow protein diffusion in and out of the porous layer of the column (Figure 1). In addition, by using a large (3.2 µm) silica core, the resultant particle is 3.6 µm in diameter which allows for longer columns at lower backpressures. The overall result is a column that delivers a significant improvement in separation power for intact proteins and their PTM-based impurities. An example of this improvement is shown in Figure 2, where degraded myoglobin is compared between a fully porous 3.5 µm wide pore column and the Aeris WIDEPORE 3.6 µm column. Note the dramatically increased number of resolved impurities for the core-shell Aeris column compared to the fully porous column.

An additional example where improved resolution of the core-shell Aeris WIDEPORE column leads to more accurate quantitation of impurities for biogeneric proteins is shown in Figure 3, where an intact alpha interferon sample was compared between a fully po-rous wide pore column and the core-shell Aeris WIDEPORE col-umn. While resolution is not complete between the interferon peak and modified components, one can see the additional component resolved by the Aeris WIDEPORE column leading to more accurate quantitation of the impurities present. One could potentially use a longer Aeris column and a shallower gradient to further resolve the components. Another characteristic of the Aeris WIDEPORE core-shell media compared to fully porous 300 Å media is that it is significantly less hydrophobic than most fully porous media, so proteins will tend to elute at a lower percentage organic. Thus, to improve resolution on existing protein methods, one should look to lower the initial percentage organic and potentially use shallower gradients when transferring a method to Aeris WIDEPORE core-shell columns.

An additional benefit of the low hydrophobicity of the column is better recovery of hydrophobic proteins. This, in combination with a well bonded inert surface and an optimized diffusion path, can lead to dramatic differences when compared to other fully porous 300 Å columns. An example of this behavior is shown in Figure 4 where epidermal growth factor (EGF) is compared between Aeris WIDEPORE 3.6 µm C4 and a fully porous 3.5 µm 300 Å C3 column. In this example, the difference in peak heights between the chro-matograms is mostly due to adsorption of the protein on the fully porous column. For most applications tested, Aeris WIDEPORE core-shell columns deliver higher protein recovery in addition to the improved resolution that the column demonstrates.

A final example to demonstrate the utility of Aeris WIDEPORE col-umns for intact biogeneric protein analysis is shown in Figure 5. In this example, RNase is reduced with DTT and different mixtures of the reduced and native protein are overlaid in the figure. Ana-lyzing and quantitating the folding state of a recombinant protein is primarily done by reversed phase chromatography of the intact protein. This example shows how an Aeris WIDEPORE column can easily resolve folded and unfolded forms of the RNase protein mak-ing it an ideal solution for analyzing intact proteins.

* Agilent and ZORBAX are registered trademarks of Agilent Technologies, Inc. Phenomenex in not affiliated with Agilent Technologies, Inc. Study was performed using new columns and, to the extent possible, identical experimental conditions were applied. Comparative separations may not be representative of all applications.

For additional technical notes, visit www.phenomenex.com

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TN-1111

Figure 3. Comparison between the Aeris WIDEPORE 3.6 µm XB-C8 and a 300 Å fully porous 3.5 µm C8 column. Interferon alpha-2a was used to compare the two columns. Note the additional impurity partially resolved by the Aeris WIDEPORE core-shell column. In both cases a 150 x 4.6 mm column was used. Flow rate was 1 mL/min and the gradient was from 30 to 65 % B in 30 minutes. One could potentially use a longer Aeris column (250 x 4.6 mm) and a shallower gradient to improve resolution of the impurities.

Column: Aeris™ WIDEPORE 3.6 µm XB-C8Fully Porous 300 Å 3.5 µm C8

Dimensions: 150 x 4.6 mmMobile Phase: A: Water with 0.1 % TFA

B: Acetonitrile with 0.1 % TFAGradient: A/B (70:30) to A/B (35:65) over 30 min

Flow Rate: 1.0 mL/minTemperature: 22 ºC

Injection Volume: 5 µLInstrument: Agilent® 1200

Detection: UV @ 214 nm (ambient)Sample: Interferon alpha-2a

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AppID 19828

Fully Porous 300 Å 3.5 µm C8

App

ID 1

9828

Aeris WIDEPORE 3.6 µm XB-C8

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App

ID 1

9827

Aeris WIDEPORE 3.6 µm C4

Column: Aeris WIDEPORE 3.6 µm C4 ZORBAX 300SB 3.5 µm C3

Dimensions: 150 x 2.1 mmMobile Phase: A: Water with 0.1 % TFA

B: Acetonitrile with 0.1 % TFAGradient: A/B (97:3) for 3 min to A/B (35:65) over 45 min



Detection: UV @ 214 nm (ambient)Sample: Human Epidermal Growth Factor (EGF)

Figure 4. Comparison between the Aeris WIDEPORE 3.6 µm C4 and a 300 Å fully porous 3.5 µm C3 column. Recombinant EGF was used to compare the two columns. Note the dramatic increase in recovery for the Aeris column and the multiple components present in the sample. In both cases a 150 x 2.1 mm column at a temperature of 40 °C was used. Flow rate was 0.3 mL/min and the gradient was from 3 to 65 % B in 45 minutes.

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ID 1

9839

*Agilent® ZORBAX® 300SB 3.5 µm C3

Protein Adsorbed

Column: Aeris WIDEPORE 3.6 µm C4Dimensions: 150 x 4.6 mm

Part No.: 00F-4486-E0Mobile Phase: A: Water with 0.1 % TFA

B: Acetonitrile with 0.1 % TFAGradient: A/B (97:3) for 3 min to A/B (35:65) over 30 min



Detection: UV @ 214 nm (ambient)Sample: RNase subject to reduction

100 % intact20 % reduced40 % reduced60 % reduced 100 % reduced

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Figure 5. RNase refold assay on Aeris WIDEPORE 3.6 µm C4. Overlays of different mixtures of reduced and non-reduced RNase are shown on an Aeris 150 x 4.6 mm column. Flow rate was 1.2 mL/min and column was held at ambi-ent temperature. The gradient was from 3 to 65 % B in 30 minutes after a 3 minute hold.

App

ID 1

9838

App

ID 1

9883

* Agilent and ZORBAX are registered trademarks of Agilent Technologies, Inc. Phenomenex in not affiliated with Agilent Technologies, Inc. Study was performed using new columns and, to the extent possible, identical experimental conditions were applied. Comparative separations may not be representative of all applications.

11

TN-1111

If Aeris core-shell technology does not provide at least an equiva-lent separation as compared to other products of the same phase and dimensions, return the product with comparative data within 45 days for a FULL REFUND.


Trademarks Agilent and ZORBAX are registered trademarks of Agilent Technologies, Inc. Aeris is a trademark of Phenomenex, Inc. Kinetex is a registered trademark Phenomenex in the United States, European Union, and other jurisdictions.



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ConclusionMaximizing resolution between proteins and their modifiedimpurities is critical in obtaining useful quantitation of post-translational modifications of biogeneric proteins. The different applications in this technical note show the utility of Aeris WIDEPORE columns for obtaining accurate data for intact protein applications. The optimized geometry of the core-shell Aeris WIDEPORE columns, as well as good selectivities of the three separate phases offered (XB-C18, XB-C8 and C4), deliver better resolution and recovery than existing fully porous 300 Å columns for intact protein analysis. Finally, the large particle size of the Aeris WIDEPORE column delivers a significantly lower backpressure than sub-2 µm 300 Å columns which allows for more flexibility in instrument used (HPLC or UHPLC) as well as column length in developing biogeneric protein applications.

Aeris™ WIDEPORE 3.6 µm Minibore Columns (mm)50 x 2.1 100 x 2.1 150 x 2.1 250 x 2.1

XB-C18 00B-4482-AN 00D-4482-AN 00F-4482-AN 00G-4482-ANXB-C8 00B-4481-AN 00D-4481-AN 00F-4481-AN 00G-4481-ANC4 00B-4486-AN 00D-4486-AN 00F-4486-AN 00G-4486-AN

Aeris WIDEPORE 3.6 µm Analytical Columns (mm)100 x 4.6 150 x 4.6 250 x 4.6

XB-C18 00D-4482-E0 00F-4482-E0 00G-4482-E0XB-C8 00D-4481-E0 00F-4481-E0 00G-4481-E0C4 00D-4486-E0 00F-4486-E0 00G-4486-E0

For more information on Aeris Core-Shell HPLC | UHPLC columns visit www.phenomenex.com/Aeris


Fully Porous 300 Å 3.5 µm C8


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TN-1124

Using Longer Aeris™ PEPTIDE Core-Shell HPLC/UHPLC Columns for Improved Peptide MappingMichael McGinley, Deborah Jarrett, and Jeff Layne Phenomenex, Inc., 411 Madrid Avenue, Torrance, CA 90501 USA

ConclusionMaximizing resolution between proteins and their modified impurities is critical in obtaining useful quantitation of post-trans-lational modifications of biogeneric proteins. The different appli-cations in this technical note show the utility of Aeris WIDEPORE columns for obtaining accurate data for intact protein applications. The optimized geometry of the core-shell Aeris WIDEPORE col-umns, as well as good selectivities of the three separate phases offered (XB-C18, XB-C8 and C4), deliver better resolution and re-covery than existing fully porous 300 Å columns for intact protein analysis. Finally, the large particle size of the Aeris WIDEPORE column delivers a significantly lower backpressure than sub-2 µm 300 Å columns which allows for more flexibility in instrument used (HPLC or UHPLC) as well as column length in developing biogene-ric protein applications.

A new 3.6 µm 100 Å HPLC/UHPLC column (Aeris PEPTIDE) has been introduced that is specifically designed to improve separations of peptide and peptide mapping applications. The Aeris PEPTIDE XB-C18 column was developed to complement Aeris WIDEPORE XB-C18 core-shell columns for protein characterization. When one looks at peptide mapping applications, performance requirements are significantly different versus intact protein separations, as increased retention and selectivity are required to separate the large number of peptides generated in peptide mapping applications. Because increased resolution is a higher priority versus speed, a larger particle (3.6 µm) core-shell particle was developed allowing the use of longer columns at lower backpressures. In this application the increased resolution that longer Aeris PEPTIDE 3.6 µm XB-C18 provide will be demonstrated.

Materials and MethodsAll chemicals, standards and antibodies were obtained from Sigma Chemical (St. Louis, Missouri). Solvents were purchased from EMD (San Diego, California). Core-shell Aeris PEPTIDE 3.6 µm XB-C18 columns (150 × 4.6 mm and 250 × 4.6 mm) were obtained from Phenomenex (Torrance, California). Bovine serum albumin was digested with trypsin and analyzed on an Agilent 1200 HPLC system with autosampler, column oven, solvent degasser, and UV detector set at 214 nm. Data was collected using ChemStation software (Agilent, Santa Clara, California). Mobile phases used were 0.1 % Formic acid in water (A) and 0.1 % Formic acid in acetonitrile (B) with a gradient from 3 to 65 % B at a flow rate of 1.2 mL/min. Gradient times were adjusted based on column length (33 to 55 minutes respectively). Column was maintained at 40 °C.

Results and DiscussionAeris PEPTIDE 3.6 µm XB-C18 core-shell particles demonstrate similar or better performance than sub-2 µm fully-porous col-umns at a fraction of the backpressure, allowing the use of longer columns at backpressures compatible with existing HPLC sys-tems. The 3.6 µm core-shell media is of particular utility for pep-tide map applications where the increased resolution of longer columns is desired (for high-speed UHPLC applications the Aeris PEPTIDE 1.7 µm XB-C18 can be used instead). An example of the utility is demonstrated in Figure 1 where 150 × 4.6 mm and 250 × 4.6 mm Aeris PEPTIDE 3.6 µm XB-C18 columns were compared for a peptide map of BSA. The 150 × 4.6 mm column provides excellent separation of the peptide mixture at a low column back-pressure (140 bar at 1.2 mL/min) such that a longer column could be used to achieve additional resolution if required. When the 250 × 4.6 mm Aeris PEPTIDE 3.6 µm XB-C18 column was used for the separation, additional peptides were resolved while still at a backpressure amenable to using standard HPLC systems (200 bar at 1.2 mL/min). These results demonstrate the perfor-mance advantage and utility of the Aeris PEPTIDE 3.6 µm XB-C18 media for highly complex peptide mapping mixtures where one can utilize different column lengths to optimize resolution and separation time based on the needs of a specific application.

Figure 1. BSA Tryptic map separated on different length Aeris PEPTIDE 3.6 µm XB-C18 columns (150 × 4.6 mm top, 250 × 4.6 mm bottom). Note the good separation on the shorter Aeris PEPTIDE column and the increased resolution provided by the longer Aeris PEPTIDE (250 × 4.6 mm) column. Because backpressure for the Aeris 3.6 µm column is so low, one can optimize column lengths based on their separation time and resolution requirements.

7 8 9 10 11 12 min

10

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mAU

19880

App

ID 1

9880

12 14 16 18 20 22 min

20

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19882

mAU

App

ID 1

9882

13

TN-1124

If Aeris core-schell columns do not provide at least an equiva-lent separation as compared to a competing column of the same phase, return the column with the comparative data within 45 days for a FULL REFUND.


Trademarks Aeris and SecurityGuard are registered trademarks Phenomenex.

Disclaimer Comparative separations may not be representative of all applications.


SecurityGuard™ ULTRA Cartridge Holder (for 2.1 to 4.6 mm ID columns)SecurityGuard ULTRA Guard Cartridge Holder ea

AJ0-9000

Aeris PEPTIDE 1.7 µm Minibore Columns (mm)SecurityGuard* ULTRA Cartridges*

50 x 2.1 100 x 2.1 150 x 2.1 3/pkXB-C18 00B-4506-AN 00D-4506-AN 00F-4506-AN AJ0-8948

Aeris PEPTIDE 3.6 µm Minibore Columns (mm)SecurityGuard ULTRA Cartridges*

50 x 2.1 100 x 2.1 150 x 2.1 250 x 2.1 3/pkXB-C18 00B-4507-AN 00D-4507-AN 00F-4507-AN 00G-4507-AN AJ0-8948

Aeris PEPTIDE 3.6 µm Analytical Columns (mm)SecurityGuard ULTRA Cartridges*

100 x 4.6 150 x 4.6 250 x 4.6 3/pkXB-C18 00D-4507-E0 00F-4507-E0 00G-4507-E0 AJ0-8946

* SecurityGuard ULTRA cartridges require holder part number, AJ0-9000

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Relevant Parameters in Developing Protein Aggregation Methods Using Yarra™ GFC ColumnsMichael McGinley, Ying Wang, and Ismail Rustamov Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

A new high efficiency GFC column, Yarra, was recently introduced and is significantly more efficient than other GFC columns on the market. In addition to higher efficiency, Yarra columns demonstrate significantly higher inertness to ionic interactions versus other GFC columns; however, such chemical characteristics sometimes require changes to operating parameters. Performing method de-velopment for protein aggregation analysis using next-generation Yarra GFC columns will be discussed.

IntroductionBeing an isocratic method, gel filtration chromatography is as-sumed to be a simple method with little or no method develop-ment involved. On closer inspection, however, subtle changes in mobile phase and other parameters can have significant results on separation performance and accuracy of determining the ag-gregation state of a protein. Protein gel filtration columns are typi-cally made by bonding a highly polar “diol-like” ligand to a porous silica matrix of a specific pore size. This polar “diol” coat on the silica is intended to minimize surface interactions between the silica and proteins, resulting in separations based on the size of a protein in solution as proteins are differentially excluded from the pores of the silica particle. One typically uses different pore-sized columns that provide maximum resolution of a specific molecular weight range based on the protein being separated. Often, two different pore-sized columns overlap in a molecular weight range, resulting in different selectivities based on the column being used.Sometimes a column at one edge of the overlap demonstrates better resolution in a protein specific manner. Since protein sep-arations are typically looking at trying to quantitate non-covalent aggregates in solution, it is most common that a buffered aque-ous mobile phase is used for GFC separations.

While coating GFC silica with a polar bonded phase greatly re-duces the amount secondary interactions between proteins and the silica matrix, the reality is that some secondary interaction persists. The secondary interactions can be summed up into two categories: ionic interactions between acidic free silanol groups on the silica surface and basic residues of a protein, and hydro-phobic interactions between the bonded phase ligand and hydro-phobic pockets of a protein. Especially aggregated proteins tend to be more sensitive to hydrophobic and ionic interactions. De-pending on the protein and column being used, different running conditions can be utilized to minimize such secondary interaction, providing for more accurate quantitation of low level impurities in a protein product. Examples will show how mobile phase compo-sition and column selection can have a major impact on separa-tions and factors to consider in developing a gel filtration method using the new Yarra brand of GFC columns.

Material and MethodsStandard proteins and mobile phase modifiers were purchased from Sigma Chemicals (St. Louis, MO, USA). Additional test proteins were purchased from R+D systems (Minneapolis, MN, USA). Mobile phases were obtained from EMD (San Diego, CA, USA). The instrument used for all separations was an Agilent® 1200 HPLC with an autosampler, column oven, and multi-wave-length detector. All separations used Yarra SEC-2000 and Yarra

SEC-3000 columns (300 x 7.8 mm) obtained from Phenomenex (Torrance, CA, USA). Mobile phases with varying molarities of phosphate and sodium chloride were used (noted in each chro-matogram).

Results and DiscussionColumn Selection

Gel filtration chromatography separates proteins based on their size in solution and how much a particular protein can perme-ate into the pore of the bonded silica particle used in the column packing. Larger proteins will permeate a decreased distance into the porous particle versus smaller proteins which will permeate deeper into the particle. As a result, fully excluded aggregates and large proteins will elute first, followed by multimers, then the full-length monomer protein, and then protein fragments with salts and small molecules coming out last. Each specific Yarra column has an optimal molecular weight separation range which is based on the pore size of the porous media: Yarra SEC-4000 (500 Å), Yarra SEC-3000 (290 Å) and Yarra SEC-2000 (145 Å). Figure 1 lists the suggested molecular weight range of each media based on the mobile phase conditions used. Note that there is significant molecular weight overlap between each in-dividual phase. Depending on the separation goal of a specific GFC application, one or the other phase may be appropriate.

While the focus is more on buffer influences, comparing Figure 2 and Figure 3 demonstrates that in overlap region, application goals may influence column choice. The separation between Ig-G dimer and monomer (Rs 1,2) is slightly better on the Yarra SEC-3000 while resolution between Ig-G monomer and BSA (Rs 2,3) is slightly better on the Yarra SEC-2000. For any overlapping mo-lecular weight region, both columns should be evaluated for that application.

Figure 1. Table of suggested separation ranges for particular Yarra SEC columns based on a specific class of mobile phase used for the separation (native or denaturing). Note that the three phases (SEC-2000, SEC-3000, and SEC-4000) all overlap under all conditions; in many cases more than one phase might be appropriate for a separation.

Molecular Weight (MW) Separation Ranges for Yarra

102 103 104 105 106 107

Molecular Weight (Daltons)

40003000

2000

40003000

2000

40003000

2000

Under

6 M GnHCI

Under

0.5 % SDS

Native

5,000 - 700,0001,000 - 150,000

500 - 100,000

15,000 - 1,500,0005,000 - 700,000

1,000 - 300,000

15,000 - 500,0005,000 - 100,000

200 - 75,000

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Buffer Influence on GFCWhile gel filtration columns are manufactured to minimize any secondary interactions between stationary phase and proteins of interest, both ionic and hydrophobic interactions do occur which can lead to peak tailing, loss of resolution, and poor recovery. Protein structure can be sensitive to changes in pH and osmolar-ity resulting in changes to net charge, solubility, and overall size based on the buffer conditions present in a protein solution. As a result, mobile phase can have a major impact on protein sepa-rations by GFC and is the main method for optimizing a particu-lar separation. Mobile phase pH can have a significant effect on chromatography due to changes in the net polarity of a protein; even a change as small as 0.2 pH units can have an impact on a separation and should be investigated whenever practical. How-ever, for most native GFC applications mobile phase pH will gen-erally be between pH 6.5 to pH 7.5.

Figure 2: An overlay of an Ig-G and BSA mixture run on a Yarra SEC-2000 column using different mobile phases. Figure 2a shows the full chromatogram and buffer conditions. Figure 2b is a zoom-in of the main components in the mixture. Note that resolution, peak shape and retention all change with mobile phase concentrations and that optimal resolution between Ig-G and BSA is obtained with moderate salt concentration on the Yarra SEC-2000 column demonstrating the utility of testing multiple mobile phase conditions

Buffer molarities and solution osmolarity can have the biggest impact on a protein separation because of its joint influence on both the protein and stationary phase. Some typical examples of this are shown in Figure 2 and Figure 3 for an Ig-G/ BSA mix-ture run on the Yarra SEC-2000 and Yarra SEC-3000, respective-ly. While there are multiple interactions occurring, increasing the buffer or salt concentration of a mobile phase influences the sec-ondary interactions between analytes and the stationary phase.

Figure 3: An overlay of the same Ig-G and BSA mixture run on a Yar-ra SEC-3000 column using different mobile phases. Figure 3a shows the full chromatogram and buffer conditions. Figure 3b is a zoom-in of the main components in the mixture. Note that resolution, peak shape, and retention all change with mobile phase concentrations. Note that resolu-tion between Ig-G and BSA is better for the Yarra SEC-2000 column while resolution between the monomer and dimer Ig-G peaks is better for the Yarra SEC-3000 column at low salt conditions.

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Figure 2a. Ig-G and BSA on Yarra™ SEC-2000

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Figure 2b. Ig-G and BSA on Yarra SEC-2000 (zoom in)

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Conditions for both figures:Column: Yarra 3 µm SEC-2000

Dimension: 300 x 7.8 mmPart No.: 00H-4512-KO

Mobile Phase: (see figures)Flow Rate: 1 mL/minDetection: UV @ 214 nm

Temp: AmbientInjection: 5 µL Sample: 1. IgG dimer 300 kDa

2. IgG (0.285 mg/mL) 150 kDa3. BSA (0.2 mg/mL) 66 kDa

Conditions for both figures:Column: Yarra 3 µm SEC-3000

Dimension: 300 x 7.8 mmPart No.: 00H-4513-KO

Mobile Phase: (see figures)Flow Rate: 1 mL/minDetection: UV @ 214 nm

Temp: AmbientInjection: 5 µL Sample: 1. IgG dimer 300 kDa

2. IgG (0.285 mg/mL) 150 kDa3. BSA (0.2 mg/mL) 66 kDa

Figure 3b. Ig-G and BSA on Yarra SEC-3000 (zoom in)

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Increasing salt concentration of the mobile phase suppress-es ionic interactions between the stationary phase and proteins which can result in better peak shape for basic proteins. How-ever, increasing salt leads to increased hydrophobic interactions between the bonded stationary phase and hydrophobic protein resulting in increased anomalous retention, peak tailing, and re-duced recovery. This is best shown in Figure 4 where IGF-1, a small moderately hydrophobic protein, is injected on a Yarra™ SEC-2000. While recovery is not adversely affected, note that as salt increases in the mobile phase the peak shape and efficien-cy is adversely affected. Retention also increases slightly for the monomer that positively influences resolution between the dimer and monomer peak, but negatively influences resolution between the monomer peak and low molecular weight impurities. Thus, for specific protein aggregate applications investigating optimal buffer concentration is suggested. Good starting points for any method would be starting with phosphate (or tris) buffer between the 50-150 mM range and then investigating if additional salt (up to 300 mM NaCl) or organic (up to 10 % of either acetonitrile or methanol) improves a separation.

Figure 4: An overlay of different mobile phase conditions for IGF-1 on Yarra SEC-2000. Note the different resolutions of particular critical com-ponents under the various salt concentrations. Low molecular weight res-olution decreases with increasing salt while dimer/monomer resolution increases. Optimizing mobile phase is critical for any GFC separation.

ConclusionBecause GFC is an isocratic separation method based on dif-ferential pore exclusion, method development considerations are much different than what is typically used for other sep-aration modes. Column selection is the main parameter con-sidered in developing a GFC method where different pore size columns determine the molecular weight range of a separa-tion. In the case of the Yarra brand of GFC columns, the Yarra SEC-2000 column is most appropriate for a lower molecular weight range of separations (1-150 kDa) and the Yarra SEC-4000 column is most appropriate for the largest molecular weight range of separations (300-1000 kDa). The Yarra SEC-3000 column is most appropriate for separations between the other two phases (between 50-500 kDa) with significant mo-lecular weight overlap between the three phases. This overlap makes it highly advantageous to investigate two phases when overlap occurs to determine which column provides the opti-mal separation for a particular application.

The most overlooked area of method development for GFC phases is the mobile phase buffer used for a separation. In-creasing or decreasing buffer and salt concentration can ei-ther increase or decrease secondary interactions between a protein and the stationary GFC phase. Increasing salt and buf-fer concentrations reduce ionic interactions possibly improv-ing separation of basic and fairly polar proteins but come with a concomitant increase in hydrophobic interactions. Reducing salt concentrations reduces hydrophobic interactions but in-creases ionic interactions. As Yarra is very inert to compared to other GFC columns, optimal salt and buffer concentrations will generally be lower than other GFC methods. The key to any GFC separation is using the optimized phase and mobile phase conditions to achieve ones separation goals.

Column: Yarra 3 µm SEC-2000Dimension: 300 x 7.8 mm

Part No.: 00H-4512-KOMobile Phase: (see figure)

Flow Rate: 1 mL/minDetection: UV @ 214 nm

Temp: AmbientInjection: 5 µL Sample: IGF-1

IGF-1 on Yarra SEC-2000





Rs 2.95

Rs 2.92

Rs 3.14

Rs 3.22

Rs 6.43

Rs 6.79

Rs 5.68

Rs 5.69

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Terms and Conditions Subject to Phenomenex Standard Terms and Conditions which may be viewed at www.phenomenex.com/TermsAndConditions.

Trademarks Yarra and SecurityGuard are trademarks of Phenomenex.

Disclaimer Comparative separations may not be representative of all applications. SecurityGuard is patented by Phenomenex. U.S. Patent No. 6,162,362

The opinions stated herein are solely those of the speaker and not necessarily those of any company or organization.


Yarra™ 3 µm SEC Columns (mm)Narrow Bore Analytical SecurityGuard™ Cartridges (mm)

Phases 300 x 4.6 300 x 7.8 4 x 3.0*

Yarra 3 µm SEC-2000 00H-4512-E0 00H-4512-K0 AJ0-4487

Yarra 3 µm SEC-3000 00H-4513-E0 00H-4513-K0 AJ0-4488

Yarra 3 µm SEC-4000 – 00H-4514-K0 AJ0-4489

for ID 4.6 - 7.8 mm

*SecurityGuard Analytical cartridges require holder, Part No.: KJ0-4282


If Yarra analytical columns do not provide at least an equivalent or better separation as compared to competing column with similar dimension, phase, and dimensions, return the column with com-parative data within 45 days for a FULL REFUND.

Better Size Exclusion, Even Better Price

www.phenomenex.com/Yarra

Aqueous Size Exclusion Columns

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Better Gel Filtration: A New Look at Method Development Parameters Using Yarra™ GFC Columns Michael McGinley, Ismail Rustamov, Michael Klein, et al. Phenomenex, Inc., 411 Madrid Ave., Torrance, CA 90501 USA

An affordable, high efficiency Yarra GFC column was recently in-troduced and is significantly more efficient than other GFC col-umns on the market. In addition to higher efficiency, Yarra columns demonstrate significantly higher inertness to ionic interactions versus other GFC columns; however, such chemical characteris-tics require optimization to operating parameters. In particular, salt and buffer concentration can have a significant impact on second-ary interactions which can influence GFC separations. Performing method development through mobile phase composition using next-generation Yarra GFC columns will be discussed.

IntroductionGel filtration chromatography (GFC) is a technique focused on separating proteins based on their size in solution (which directly correlates to their molecular weight). This makes GFC particularly useful for quantitating protein aggregation state as well as post translational modifications that impact solution structure.1 De-spite being a seemingly simple isocratic separation method there are several critical parameters in optimizing separation methods for specific proteins. Buffer and salt concentrations of the mobile phase can have a significant impact on the retention and resolu-tion of many proteins (especially basic and hydrophobic proteins) and optimization strategies for GFC will be discussed.2

Materials and MethodsGFC standards using various proteins and void markers (thyro-globin, IgA, IgG, ovalbumin, myoglobin, lysozyme, and uridine; Sigma Chemical, St. Louis, MO, USA) were used to evaluate sec-ondary interactions of the stationary phase as well as determine molecular weight linearity. Isocratic HPLC runs were performed

on an Agilent® 1100 with autosampler, UV detector, and ChemSta-tion™ software (Agilent Technologies, Palo Alto, CA, USA). HPLC columns used for GFC studies were Yarra 3 µm SEC-2000 and Yarra 3 µm SEC-3000 (300 x 7.8 mm dimension; Phenomenex, Torrance, CA, USA). Columns were run at ambient temperature and at a flow rate of 1.0 mL/min unless otherwise noted. Mobile phase buffer composition is listed in specific figures.

Results and DiscussionEfforts were undertaken to show influences that mobile phase can have on GFC separations in order to give researchers a better understanding on method development actions to take in de-veloping methods. Although GFC bonded stationary phases are designed to minimize interactions between negatively-charged silanol groups on the silica surface and basic proteins, some in-teraction does occur and that can be minimized by increasing salt concentration in the mobile phase thus reducing ionic interac-tions. An excellent example of this is shown in Figure 1 where ly-sozyme (a basic protein) is injected on a Yarra SEC-3000 column under different mobile phase concentrations. Note that as the salt concentration in the mobile phase increases, the retention time of lyzosyme decreases to the appropriate retention for a protein of its molecular weight. In addition, both recovery and peak shape improves as salt concentration of the mobile phase increase. Such results would suggest that increasing salt concentration is the key for all GFC separations. Unfortunately that is not the case as ionic interactions are only one of the interactions that occur during GFC that can impact performance.

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Bonded phases used for gel filtration chromatography typically have some diol functionality. The diol ligand both covers the sili-ca surface as well as displays a polar functionality that “mimics” water. However, besides GFC, diol phases are sometimes used as very weak hydrophobic interaction media; thus one would ex-pect that at increased salt concentrations in the mobile phase one would start seeing hydrophobic interactions occurring be-tween the bonded phase and hydrophobic proteins. To test this hypothesis, a mixture of standard proteins was run on a Yar-ra™ SEC-3000 column and salt concentration was increased. Figure 2 compares the retention of ovalbumin (a moderately hy-drophobic protein) to other proteins as the salt concentration in the mobile phase increases. One can see ovalbumin increase in retention to a molecular weight anomalous to the expected mo-lecular weight. Note that most of the other proteins only change minimally in retention time versus ovalbumin. This difference can be better visualized when one makes a plot of retention time ver-sus the log of the molecular weight for the proteins in the mixture. The log plot in Figure 3 for this experiment shows that only oval-bumin moves substantially with changes in mobile phase concen-trations of salt suggesting that for hydrophobic proteins too much salt can be detrimental to separation. Further, it suggests that method development for GFC revolves around finding the optimal mobile phase to minimize ionic and hydrophobic interactions.

To determine if this observation is specific to the GFC phase used in these experiments and mobile phase buffer salts, the experi-ment was repeated using a different pore size GFC column (Yar-ra SEC-2000) and utilizing sodium sulfate as the mobile phase salt. When ones looks at the results in Figure 4, one can see a similar result for ovalbumin as in previous figures with retention time increasing as salt concentration increases. Another partic-ular observation unique for the Yarra SEC-2000 column revolves around the thyroglobulin/aggregate peak early in the chromato-gram; under low salt conditions, thyroglobulin appears to be fully excluded, yet with other mobile phase conditions some resolu-tion is obtained. It is possible that a similar hydrophobic effect is being observed with the increased retention of the thyroglobulin peak leading to some resolution between it and a high molecular weight aggregate peak. Other proteins in the mixture do shift slightly, but not as profound as observed with ovalbumin or thyro-globulin. These results suggest that salt can influence the hydro-phobic retention of some proteins on GFC phases in general but not specific to a particular salt or column phase.

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50 mM Phosphate

Rs 1.43

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Figure 2. Yarra SEC-3000 pH 6.8

An overlay of a standard protein mixture (thyroglobulin, IgA, IgG, ovalbumin, myoglobin, and uridine) run on a Yarra SEC-3000 column with increasing salt concentration (mobile phase concentration listed with each chromatogram). Note that ovalbumin increases retention as salt increases. Increasing salt concentration increases hydrophobic interactions between the bonded diol phase and some protein analytes.

Ap

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Column: Yarra 3 µm SEC-3000Dimensions: 300 x 7.8 mm

Part No.: 00H-4513-K0Mobile Phase: 50 mM Sodium phosphate pH 6.8 + Sodium

chloride (see figure for concentration)Flow Rate: 1 mL/minDetection: UV @ 280 nmInjection: 5 µLSample: 1. Thyroglobin 699 kDa

2. IgA 300 kDa3. IgG 150 kDa4. Ovalbumin 44 kDa5. Myoglobin 17 kDa6. Uridine


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References1. J. Engelsman; P. Garidel; R. Smulders; H. Koll; B. Smith; S. Bassarab;

A.Seidl; O. Hainzl; W. Jiskoot; Pharm Res (2011) 28:920–933

2. T. Arakawa; D. Ejima; T. Li; J. Philo; Journal of Pharmaceutical Sciences V99 (4) pg 1674–1692 (2010

ConclusionGel filtration chromatography is an isocratic method for sepa-rating proteins based on their size in solution, but secondary interactions between the stationary phase and protein ana-lytes make it far from simple. Mobile phase composition plays a significant role in the separation of protein by influencing proteins in solution as well as the ionic and hydrophobic in-teractions that the stationary phase may impact on the sep-aration. While not studied in this paper, mobile phase pH can influence the net charge of a protein as well as the surface silanols on the silica media. The use of buffer (and non-buffer) salts in the mobile phase can dramatically influence the re-

tention, peak shape, and recovery of proteins by modulating ionic secondary interactions. While increasing salt concentr-tion reduces ionic interaction, it also increases hydrophobic interactions making method development for GFC separation a balance between the two interactions. The key to optimizing any GFC method is investigating a range of salt concentra-tions and compositions to achieve the desired separation of the proteins or other biomolecules of interest.

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Overlay of the standard protein mixture on a Yarra SEC-2000 column. In this figure, the standard used in Figure 2 was injected on a smaller pore size GFC column (Yarra SEC-2000) using a different salt (sodium sulfate). Similar retention shifts for ovalbumin were observed indicating that this effect is not salt or column dependent.

Figure 4. Yarra SEC-2000 pH 6.8

Column: Yarra 3 µm SEC-2000Dimensions: 300 x 7.8 mm

Part No.: 00H-4512-K0Mobile Phase: 50 mM Sodium phosphate pH 6.8 + Sodium

sulfate (see figure for concentration)Flow Rate: 1 mL/minDetection: UV @ 280 nmInjection: 5 µLSample: 1. Thyroglobin 699 kDa

2. IgA 300 kDa3. IgG 150 kDa4. Ovalbumin 44 kDa5. Myoglobin 17 kDa6. Uridine

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Protein Mixture Calibration Curve on Yarra SEC-3000

1000000

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t 50 mM Phosphate

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l 200 mM NaCl + 50 mM Phosphate

ª 300 mM NaCl + 50 mM Phosphate

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A log molecular weight plot for the protein standards shown in Figure 2 under different mobile phase conditions. Most proteins demonstrate limited reten-tion change except for ovalbumin which shifts to greater retention with increased salt.

Figure 3. Protein Mixture Calibration Curve on Yarra SEC-3000

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Terms and Conditions Subject to Phenomenex Standard Terms and Conditions which may be viewed at www.phenomenex.com/TermsAndConditions.

Trademarks Yarra and SecurityGuard are trademarks of Phenomenex. Agilent is a registered trademark and ChemStation is a trademark of Agilent Technologies, Inc.

Disclaimer Comparative separations may not be representative of all applications

Phenomenex is not affliated with Agilent.

SecurityGuard is patented by Phenomenex. U.S. Patent No. 6,162,362



Yarra™ 3 µm SEC Columns (mm)

Narrow Bore Analytical AnalyticalSecurityGuard™ Cartridges (mm)

Phases 300 x 4.6 150 x 7.8 300 x 7.8 4 x 3.0*

/10 pk

Yarra 3 µm SEC-2000 00H-4512-E0 00F-4512-K0 00H-4512-K0 AJ0-4487

Yarra 3 µm SEC-3000 00H-4513-E0 00F-4513-K0 00H-4513-K0 AJ0-4488

Yarra 3 µm SEC-4000 00H-4514-E0 – 00H-4514-K0 AJ0-4489

for ID 4.6 - 7.8 mm*SecurityGuard Analytical cartridges require holder, Part No.: KJ0-4282

SecurityGuard Cartridge Holder Kit

Part No. Description

KJ0-4282 for 4.6 to 7.8 mm ID column

If Yarra analytical columns do not provide at least an equivalent or better separation as compared to competing column with similar dimension, phase, and dimensions, return the column with comparative data within 45 days for a FULL REFUND.


Aqueous Size Exclusion ColumnsUltra-High Resolution at an Unbelievable Price

www.phenomenex.comPhenomenex products are available worldwide. For the distributor in your country, contact Phenomenex USA, International Department at [email protected]

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