Narrow Bore Gel Filtration Columns for Protein and Aggregate Analysis

By: Roy Eksteen, Reporter US Volume 31.1

Introduction

Traditionally, in Gel Filtration Chromatography (GFC), the column diameter has been larger than the diameter of HPLC columns used in interactive modes of liquid chromatography. The main reason for this was that HPLC instruments were optimized for 4.6 mm I.D. columns that were 25 cm long. To prevent extra-column band broadening (ECBB) detracting from the efficiency of the column, it was (and still is) accepted practice to retain the solute of interest at least up to a retention factor of 2; which means a peak volume that is three times the volume in which an unretained (low molecular weight) solute elutes from a GFC column. The most popular internal diameter of GFC columns is 7.8 mm. Since peak volume is proportional to the ratio of the square of the column diameters, the volume in a 7.8 mm I.D. column is almost 3 times larger than that of a 4.6 mm I.D. column. In other words, extra-column band broadening was not a factor when 7.8 mm I.D. GFC columns were used with conventional HPLC instrumentation.

In recent years, HPLC and UHPLC instruments have been optimized to take advantage of higher column efficiencies provided by newer particle technologies - sub-2 µm porous particles and superficially porous particles. When using such optimized instrumentation, the researcher can now take advantage of the primary benefit of smaller I.D. (gel filtration) columns, namely the ability to detect proteins at higher sensitivity when limited in sample mass. In this summary review of the poster presented at ISPPP 20071, we will illustrate this principle featuring the popular TSKgel® SuperSW3000 gel filtration columns.

Experimental

Columns

  • TSKgel SuperSW3000, 4 µm, 1 mm I.D. x 30 cm (Product No. 821485)
  • TSKgel SuperSW3000, 4 µm, 2 mm I.D. x 30 cm (Product No. 821845)
  • TSKgel SuperSW3000, 4 µm, 4.6 mm I.D. x 30 cm (Product No. 818675)

Sample
Proteins and enzymes were purchased from Sigma-Aldrich®. The antibody was a gift from the Tosoh Research Center (Kanagawa, Japan).

Other

  • UV cell: 2 µL (for 2 mm & 4.6 mm I.D.)
  • UV cell: 35 nL (for 1 mm I.D.)
  • Sample injector: Rheodyne 7520
  • Tubing (injector to column): 0.05 mm I.D. x 20 cm fused silica

Results and Discussion

Gel filtration chromatography (size-exclusion chromatography in an aqueous mobile phase) is a powerful tool for analyzing biological polymers such as proteins, peptides, nucleic acids, and their fragments. In biopharma companies, TSKgel SW series GFC columns are now routinely and widely used in research and in the final quality control of biotherapeutic drugs. In QC, the analyst is rarely sample mass limited. However, researchers performing proteomic studies routinely rely on the ability to detect very small amounts of proteins.

Like the conventional 4.6 mm I.D. column, the 1 mm and 2 mm I.D. TSKgel SuperSW3000 columns are filled with spherical 4 µm particles chemically bonded with diol- containing functional groups. The effect of sample mass on detection sensitivity is shown in Figure 1 for a series of protein standards. The same protein mass and volume (1 µL) was injected on a 1 mm I.D., 2 mm I.D. and 4.6 mm I.D. TSKgel SuperSW3000 column. Approximately a five-fold increase in peak height of a standard protein mixture was observed when using a 2 mm I.D. x 30 cm TSKgel SuperSW3000 column compared to a 4.6 mm I.D. x 30 cm column. Clearly, the best sensitivity is obtained on the smallest bore (1 mm I.D.) column. Note that the same 2 µL UV detector cell volume was used for the 2 and 4.6 mm I.D. columns and a much smaller 35 nL cell volume for the 1 mm I.D. column.

Figure 1. Effect of Sample Mass on   Detection Sensitivity

Figure 1. Effect of Sample Mass on Detection Sensitivity
CONDITIONS: columns: TSKgel SuperSW3000, 30 cm x 4.6 mm I.D. (Product No. 818675), TSKgel SuperSW3000, 30 cm x 2 mm I.D. (Product No. 821845), TSKgel SuperSW3000, 30 cm x 1 mm I.D. (Product No. 821485); eluent: 0.1 mol/L phosphate buffer + 0.1 mol/L Na2SO4 + 0.05% NaN3 (pH 6.7); flow rate: 0.350 mL/min (4.6 mm I.D.) 0.650 mL/min (2 mm I.D.) 0.016 mL/min (1 mm I.D.); detection: UV at 280 nm; detector cell volume: 2 µL (4.6 and 2 mm I.D.) 35 nL (1 mm I.D.); temperature: 25 °C; injection volume: 1 µL

 

The same improvement in sensitivity as seen for protein standards was also evident when analyzing an IgG sample containing a low concentration of aggregates, as is shown in Figure 2. The fact that the calibration curves were linear (not shown) confirmed nonspecific adsorption on the stationary phase was minimal. The detection limit of IgG was 18 ng using the 1 mm I.D. TSKgel SuperSW3000 column while still being able to detect small amounts of IgG aggregates. As with 1 mm I.D. columns, we found that reducing the injection volume of an IgG solution from 10 µL to 1 µL greatly improved the efficiency of the 2 mm I.D. column, although at constant injection volume, efficiency did not vary with IgG concentration when in the range of 1-5 mg/mL. In a separate study (results not shown), it was determined that trace analysis of biological components was possible when the 1 mm I.D. TSKgel SuperSW3000 column was utilized with an off-line SELDI/TOF/MS.

Figure 2. Improve IgG Aggregate Detection Limit

Figure 2. Improve IgG Aggregate Detection Limit
Conditions: columns: TSKgel SuperSW3000, 30 cm x 4.6 mm I.D. (Product No. 818675), TSKgel SuperSW3000, 30 cm x 2 mm I.D. (Product No. 821845), TSKgel SuperSW3000, 30 cm x 1 mm I.D. (Product No. 821485); eluent: 0.1 mol/L phosphate buffer + 0.1 mol/L Na2SO4 + 0.05% NaN3 (pH 6.7); flow rate: 0.350 mL/min (4.6 mm I.D.) 0.650 mL/min (2 mm I.D.) 0.016 mL/min (1 mm I.D.); detection: UV at 280 nm; detector cell volume: 2 µL (4.6 and 2 mm I.D.) 35 nL (1 mm I.D.); temperature: 25 °C; injection volume: 1 µL; sample: lgG (mouse, Mab, 1 mg/mL)

 

The performance of a competitor column filled with a composite matrix of dextran and agarose is compared in Figure 3 with a 2 mm I.D. TSKgel SuperSW3000 column, each operated at their recommended flow rate. The result demonstrates the silica backbone in SW-type TSKgel columns is best suited to deliver fast and efficient results in a high throughput situation.

Figure 3. Competitive Advantage

Figure 3. Competitive Advantage
Conditions:
column: TSKgel SuperSW3000, 30 cm x 2 mm I.D. (Product No. 821845); flow rate: 65 µL/min; linear velocity: 124 cm/h; N: 30,000;
column: Competitor A, 30 cm x 3.2 mm I.D.; flow rate: 40 µL/min; linear velocity: 30 cm/h; N: 11,000;
eluent: 0.1 mol/L phosphate buffer + 0.1 mol/L Na2SO4 + 0.05% NaN3 (pH 6.7); detection: UV at 280 nm; temperature: 25 °C; injection volume: 0.2 µL

Conclusions

Narrow bore TSKgel SuperSW3000 columns (1 mm and 2 mm I.D.) showed similar resolution for biological samples to what can be obtained on conventional 4.6 mm I.D. TSKgel SuperSW3000 columns. As expected, highest sensitivity was achieved on the 1 mm I.D. narrow bore column. Linear calibration curves confirmed nonspecific adsorption on the stationary phase was minimal. The detection limit of IgG was 18 ng using a 1 mm I.D. column while still being able to detect small amounts of IgG aggregates. The results indicate narrow bore TSKgel SuperSW3000 columns are an excellent choice for the rapid separation of proteins and enzymes at micro scale and are a great fit for the trace analysis of biological components by LC/MS.

Trademarks

Fused-Core is a registered trademark of Advanced Materials Technology, Inc.
Ascentis and Sigma-Aldrich are registered trademarks of Sigma-Aldrich Co. LLC
TSKgel is a registered trademark of Tosoh Corporation

Materials

     

 References

  1. C. Tomasek, H. Moriyama, S. Yamasaki, S. Satoh and H. Tomizawa, High sensitivity protein separations using a new size exclusion chromatography microcolumn for use in conjunction with MS; ISPPP 2007, Orlando, FL, USA. View pdf containing details of this study.

 

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