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Developing SEC Methods for Biologics Analysis

Parameters such as pore size, column dimensions, temperature, flow rate, and mobile phase are important to consider when developing robust SEC methods as many of these can impact the level of aggregation detected.

Choosing the optimum pore size for your molecules of interest is very important.  You need to ensure the protein monomers and dimers are physically able to permeate the pore structure in order to obtain a separation.  One rule of thumb is that the pore size of the column should be three times the diameter of the molecules of interest.  The largest protein in Figure 1 thyroglobulin, 670 kDa, which has a hydrodynamic radius of approximately 10 nm.  IgG, 150 kDa, has a hydrodynamic radius of approximately 5 nm.  For the analysis of IgG the optimum pore size is, therefore, three times larger than the diameter of IgG, 30 nm or 300 Å. 

Figure 1. SEC separation of bovine serum albumin (BSA), immunoglobulin G (IgG), and thyroglobulin on a column with 300 Å pore size.

Figure 2 shows the same three proteins separated on a column with a pore size of 200 Å.  Clearly this is much too small for thyroglobulin and is rather small for IgG.  Notice that in this separation the IgG monomer peak, 150 kDa, is actually eluting slightly later than the BSA dimer peak, 132.8kDa, so clearly there is some non-ideal behaviour going on here and some further method development is in order.  If the pore size is too small the protein molecules will be excluded from the pores and elute in the void volume of the column, which will result in inaccurate quantitative data.  

Figure 2. SEC separation of bovine serum albumin (BSA), immunoglobulin G (IgG), and thyroglobulin on a column with 200 Å pore size.

Choosing a larger pore size than necessary may cause difficulties too; the proteins of interest will elute in a much narrower retention time window and therefore there will be less resolution (Figure 3).  If the pore size is too large all of the proteins will be able to fully permeate the particles and there will be very little separation. 
Because the choice of pore size influences the resolution obtained when using SEC it is worthwhile testing a range of pore sizes to match this to the analytes.

Figure 3. SEC separation of bovine serum albumin (BSA), immunoglobulin G (IgG), and thyroglobulin on a column with 450 Å pore size.

Column internal diameter affects the flow rate and injection volume.  There are two common column internal diameters in SEC, 4.6 and 7.8 mm.  Since the separation mechanism is purely based on diffusion into and out of the pores of the stationary phase, the greatest separation comes from having larger column sizes. 

Using a slow flow rate allows the molecules sufficient time to diffuse into and out of the static pool of mobile phase contained within the pore structure. The normal flow rate for a 7.8 mm ID SEC column is 1.0 mL/min.  This translates to 0.35 mL/min when using a smaller 4.6 mm ID column (Figure 4).  This also means the amount of sample injected on a 4.6 mm ID column can be reduced by a similar amount (~33%), which is useful if you have a limited amount of sample available. 

It is important to recognize that operating 4.6 mm ID columns at 0.35 mL/min can lead to differences in performance related to the extra-column dead volume in the system; peaks can become broader if long capillaries with wide bores are used to connect the injection valve to the column or the column to the detector.

Figure 4. Effect of column internal diameter on SEC separations.

Longer columns provide more resolution, but longer run times (Figure 5).  Shorter columns produce shorter run times, greatly increasing throughput (for even faster separations, use higher flow rates).  Since separation relies on the available pore volume, using longer columns or multiple columns in series increases the available pore volume and therefore increases resolution.  Going from a 30 cm column to a 15 cm column means the run time can be cut in half.

As long as you still have the required amount of resolution, using a shorter column can greatly improve sample throughput. This may be particularly important to you if you are screening multiple samples during early development phases, or taking regular measurements from a fermentation.

Figure 5. Effect of column length on SEC separations.

Temperature is sometimes overlooked in a simple technique such as the isocratic methods used in SEC.  Methods often state the temperature simply as ambient.  However, it is highly desirable to use a column oven if you are looking at ensuring good reproducibility.  In a laboratory environment where the ambient temperature could change more than 10 °C during the course of the day/night (factors such as weather, time of year, laboratory location can all make such differences possible), you will see a noticeable impact (Figure 6). 

This difference in temperature will change the viscosity of the mobile phase significantly.  This in turn will change the column operating pressure, and the diffusion process into and out of the pore structure will also change.  The temptation is to increase the temperature - higher temperatures will mean significantly lower viscosity, much lower operating pressures and much faster diffusion, giving sharper peaks and better resolution.  However, if the temperature is too high, more aggregation is likely to result - precipitation of the sample prior to analysis may even occur due to exposure to excessive temperature (Figure 7).

Figure 6. Effect of temperature on SEC separations.
Figure 7. Protein aggregation resulting from excessive temperature.

Chromatographers who work with reversed phase separations will be used to operating at high flow rates and achieving optimum plate counts for small molecules (e.g.  1.1-1.2 mL/min on a 300 x 7.8 mm column, Figure 8, top).  However, when you start to look at the column efficiency for larger molecules such as proteins, or even a molecule as small as Vitamin B12 (cobalamin, FW 1,355.37) you can see the optimum flow rate is much lower (in this case 0.6 mL/min compared to 1.2 mL/min for a small molecule such as uridine, Figure 8, middle). This of course means that the run times will be considerably different.

Ultimately we are interested in the resolution – the degree of separation – between our protein monomer and dimer peaks (plus higher order aggregates).  Figure 8 (bottom) illustrates the kind of resolution that may be seen for some IgG molecules, and also for some smaller proteins, and the effect that flow rate has on the resolution.  Remember, resolution is a function of the difference in retention time divided by the average peak width.
Figure 8. Effect of flow rate on SEC separations. 
Top: Optimum flow rate for small molecules. 
Middle: Optimum flow rate for biomolecules. 
Bottom: Effect of flow rate on resolution of IgG molecules.

Mobile phase selection can have a noticeable effect on some proteins; with differences in ionic strength, pH, and buffer composition resulting in changes in resolution, selectivity, and peak shape.  It is therefore essential to consider what effect even minor changes in buffer composition may have in order to demonstrate method robustness as well as method optimization.  Particular care needs to be taken with detergents and other denaturants as they can cause proteins to unfold and become larger in solution, or can bind to such an extent that molecular weight and size in solution increase dramatically leading to shorter retention times (Figure 9). 

Simply changing buffer composition (ionic strength) at constant pH shows just how some proteins may be more susceptible to differences (Figure 10). 

Adding NaCl to the composition can also result in changes (Figure 11). 

Different columns may well behave differently too; most are silica-based and it is common to see undesirable interactions occurring at low ionic strength.  However, as you increase ionic strength you may also begin to see other effects; hydrophobic interactions may start to occur as you move towards conditions that begin to look like hydrophobic interaction chromatography.

Figure 9. Effect of detergents on SEC separations.
Figure 10. Effect of sodium phosphate concentration (ionic strength) on SEC separations.
Figure 11. Effect of salt addition (e.g. NaCl) on SEC separations.

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Dr. Dawn Watson

This article was written by Dr. Dawn Watson.

Dawn received her PhD in synthetic inorganic chemistry from the University of Strathclyde, Glasgow. The focus of her PhD thesis was the synthesis and application of soft scorpionate ligands. As well as synthetic skills, this work relied on the use of a wide variety of analytical techniques, such as, NMR, mass spectrometry (MS), Raman spectroscopy, infrared spectroscopy (IR), UV-visible spectroscopy, electrochemistry, and thermogravimetric analysis.

Following her PhD she spent two years as a postdoctoral research fellow at Princeton University studying the reaction kinetics of small molecule oxidation by catalysts based on Cytochrome P450. In order to monitor these reactions stopped-flow kinetics, NMR, HPLC, GC-MS, and LC-MS techniques were utilized.

Prior to joining the Crawford Scientific and CHROMacademy technical team she worked for Gilson providing sales and support for the entire product range including, HPLC (both analytical and preparative), solid phase extraction, automated liquid handling, mass spec, pipettes, and laboratory consumables.

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