Coupling ion exchange chromatography (IEX) to multi-angle light scattering (MALS) can open new opportunities in protein characterization. Mario Lebendiker and Hadar Amartely from The Hebrew University of Jerusalem explain how.
A new analytical tool for protein characterization
MALS coupled with size exclusion chromatography (SEC) is a common method for characterizing protein mass, shape, aggregation, oligomerization, interactions, and purity. In some cases, however, the limited resolution of SEC interferes with the accurate analysis that can be achieved by MALS. For example, SEC-MALS analysis can have limitations when analyzing mixtures of protein populations with identical or very similar masses, oligomers with poor separation, and small peptides. As IEX is a separation technique with higher resolution than SEC, it has the potential to help solve some of these cases.
In our recent study, we show that combining IEX with MALS (IEX-MALS) allows a precise analysis of samples that cannot be resolved by SEC-MALS (1). The experimental work was performed on a ÄKTA pure protein purification system with a MALS detector connected via the external equipment user interface (I/O-box).
IEX-MALS is useful in these situations
The strengths of IEX-MALS analysis is particularly useful for protein samples not suitable for a typical SEC-MALS experiment. Here are some examples:
1. Oligomers with poor separation—When oligomeric species are present in the same sample, these often elute with a poor separation profile when using SEC. This leads to limited analysis by SEC-MALS without definitive and clear conclusions. When high-order oligomers or aggregates are not fully separated from the protein peak, the molecular mass determination of lower oligomers will be inaccurate. Our data includes experiments where IEX-MALS significantly improved the separation (1). This, together with the fact that lower oligomers elute before higher oligomers, facilitates a more accurate MALS analysis.
2. Proteins with very similar sizes—These proteins can’t be separated by SEC, but IEX works well if they have different pIs. An example is antibodies, which makes the technique suitable for quality control of therapeutic proteins.
3. Low molecular weight (LMW) proteins or peptides—Intensity of light scattering depends on the molar mass and the concentration of the macromolecule. Unlike SEC, there is no volume limitation in IEX. A sufficient volume of the analyzed sample is injected to the column and the target elutes highly concentrated, increasing the light scattering intensity.
4. Proteins that are difficult to concentrate—Due to the same reasons as LMW proteins. Examples are many intrinsically disorder proteins and proteins that are prone to aggregation.
For these types of proteins, the different separation principle used in IEX-MALS provides an additional and critical level of protein characterization. This can help overcome the SEC-MALS limitations described in Table 1.
Table 1. Advantages and limitations of SEC-MALS and IEX-MALS
| Method parameter | SEC-MALS | IEX-MALS |
|---|---|---|
| Principle of separation | Hydrodynamic size | Charge |
| Parameters that increase selectivity and resolution | Restricted
Different columns can be used with different fractionation range, resin particle size, matrix, or different column length. |
Varied
Different steps/gradient running programs, gradient slope, pH or salt gradient, type of salts, type of buffer, resin particle size, different matrixes, type of column and column length. |
| Injection volume | Limited | Unlimited*
Samples can be injected by loop or by pump valve: very convenient for diluted samples, difficult to concentrate proteins, or LMW proteins with low light scattering signal |
| Sample concentration | Concentrated sample | Diluted or concentrated sample |
| Sample buffer | As desired | Conditions that allow binding |
| Equilibration time | Long
From overnight to many hours. Independent runs cannot be recorded separately. |
Short
From one hour to a few minutes (we checked this for MonoQ/S and SOURCE 15Q columns) |
| Flexibility of changing parameters during the run | Not flexible | Flexible
HOLD, or change gradient slope, etc. |
| Samples can be run individually using UNICORN methods | No | Yes
The ÄKTA pure system can be stopped between runs and a clean MALS baseline is achieved with a short (5–10 min) equilibration before sample injection or during wash before elution. |
| Conjugate analysis
(for modified proteins: glycosylation, membrane proteins in micelles, etc.) achieved by using refraction index (RI) signal |
Easy to perform | More laborious: needs buffer subtraction
RI signal changes during salt or pH gradients. Requires relatively high sample concentration. With our equipment, WYATT T-rEX optilab refractometer, RI signal is unstable at > 0.5 M NaCl concentration (MALS is stable) |
| Analysis of low masses | Not recommended | Possible |
| Analysis of mixtures of proteins with similar size | Not recommended | Recommended |
* Injected volume is unlimited, although total amount of injected sample is restricted by the column capacity. For GE’s Mono Q HR 5/5 column the loading capacity is ~25 mg protein.
IEX-MALS: a great compliment to SEC-MALS
In summary, our study showed that IEX-MALS is a valuable and complementary technique for proteins where SEC-MALS analysis has limitations.
Learn more about lab-scale automation solutions that these experiments were performed on.