This protocol describes the use of high-specificity ion-exchange chromatography with multi-angle light scattering for an accurate molar mass determination of proteins, protein complexes, and peptides in a heterogeneous sample. This method is valuable for quality assessment, as well as for the characterization of native oligomers, charge variants, and mixed-protein samples.
Ion-exchange chromatography with multi-angle light scattering (IEX-MALS) is a powerful method for protein separation and characterization. The combination of the high-specificity separation technique IEX with the accurate molar mass analysis achieved by MALS allows the characterization of heterogeneous protein samples, including mixtures of oligomeric forms or protein populations, even with very similar molar masses. Therefore, IEX-MALS provides an additional level of protein characterization and is complementary to the standard size-exclusion chromatography with multi-angle light scattering (SEC-MALS) technique.
Here we describe a protocol for a basic IEX-MALS experiment and demonstrate this method on bovine serum albumin (BSA). IEX separates BSA to its oligomeric forms allowing a molar mass analysis by MALS of each individual form. Optimization of an IEX-MALS experiment is also presented and demonstrated on BSA, achieving excellent separation between BSA monomers and larger oligomers. IEX-MALS is a valuable technique for protein quality assessment since it provides both fine separation and molar mass determination of multiple protein species that exist in a sample.
Quantitative characterization of protein products is increasingly essential as a means of quality control (QC), both for regulatory purposes in the biopharmaceutical industry and to guarantee the reliability and integrity of life science research1,2. As described on the websites of protein networks Protein Production and Purification Partnership in Europe (P4EU) and Association of Resources for Biophysical Research in Europe and Molecular Biophysics in Europe (ARBRE-MOBIEU) (https://p4eu.org/protein-quality-standard-pqs and https://arbre-mobieu.eu/guidelines-on-protein-quality-control, respectively), protein QC must characterize not only the purity of the final product, but also its oligomeric state, homogeneity, identity, conformation, structure, posttranslational modifications, and other properties3,4.
One of the most common QC characterization methods is SEC-MALS. In this method, an analytical SEC column is coupled to MALS and spectrophotometric and refractometric detectors, enabling accurate measurements of the protein molar mass of each peak5. SEC-MALS determines the molar mass of the eluted peaks independently of the elution volume and overcomes the inaccuracy of the analytical SEC using column calibration. The addition of a dynamic light-scattering (DLS) module adds size measurement capability allowing hydrodynamic radius determination. In academic research, SEC-MALS is typically used to determine the oligomeric state of a protein, its conformation, the level of purity, level of aggregation, and modified proteins, such as glycoproteins or lipid-solubilized membrane proteins (determine the molar mass of protein and conjugate the components individually)6,7,8.
In many cases, the final protein of a purification process is not a well-defined molecular species but rather comprises some heterogeneity. The proteins in such a mixture can be varied in terms of structure (for example, different oligomeric forms), conformations, or protein isoforms. Protein heterogeneity can also be a result of minor chemical differences caused by C-terminal lysine processing or asparagine/glutamine deamination, leading to charge variation9,10. Differences in posttranslational modifications such as glycosylation can also lead to heterogeneous samples with charge variations9. These different types of heterogeneity are reflected in the protein’s biophysical characteristics and can impact the stability and biological activity of the target protein11.
Reliable quality control assays of such heterogeneous samples require a highly resolutive analytical separation technique. There are cases where good separation can be challenged to achieve with analytical SEC columns, due to their limited resolution and separation abilities12, resulting in flawed SEC-MALS analysis. Combining a high-specificity separation technique such as IEX with MALS can overcome the limitation of SEC-MALS in heterogeneous samples and provide a complementary method for protein characterization (Table 1 in Amartely et al.12). Unlike SEC, which separates macromolecules by their hydrodynamic size13, IEX separates macromolecules by their surface charge14. Anion exchange (AIEX) and cation exchange (CIEX) matrixes bind negatively and positively charged variants, respectively. With a fine separation between protein populations that share a relatively close mass or shape, IEX-MALS successfully determines the molar mass of each individual protein state in a mixture sample12.
Here we present a standard protocol for running an IEX-MALS experiment for the separation and analysis of BSA oligomeric forms that exist in the same sample. The choice of an IEX column for a specific protein is important and discussed, as well as the pH and conductivity conditions of the buffers. The analysis of IEX-MALS experimental data is also described step by step. Although the separation of BSA oligomers is good and sufficient in SEC-MALS, BSA is a good example to show the IEX-MALS capabilities and to demonstrate the optimization of an experiment. Examples of poor separation achieved by SEC-MALS and proper separation and analysis enabled by IEX-MALS are discussed in a previous study12.
1. Preparation of the system
2. Preparation of the sample and buffer
3. Choice and development of an IEX method for a protein
4. IEX-MALS experiment
5. Analysis of IEX-MALS experimental data
BSA is a common protein which is used in chromatography for calibration of the experimental system22 and is highly suitable for practicing IEX-MALS, as well as SEC-MALS. It is primarily monomeric with a theoretical monomer mass of 66.7 kDa and usually incorporates a small number of dimers and higher oligomers23.
BSA was analyzed on IEX-MALS using an anion exchange analytical column (see Table of Materials). A wide linear gradient consisting of 30 CV from 75 mM to 350 mM NaCl separated BSA monomers from the higher oligomers. Downstream MALS analysis resulted in a calculated monomer molar mass of 66.8 ± 0.7 kDa and in a calculated dimer molar mass of 130 ± 5 kDa (Figure 1A).
Based on the buffer conductivity at the eluted peaks, the gradient was changed to a different program: a long step of 175 mM NaCl followed by a linear gradient from 175 mM to 500 mM NaCl. The new gradient greatly improved the resolution and excellent separation between BSA monomer (with a calculated molar mass of 66.1 ± 0.7 kDa) and its higher oligomeric species (with a calculated average mass of 132 ± 2 kDa) (Figure 1B). In order to focus also on the high oligomeric species and to calculate molar masses of each individual oligomeric form of BSA, a stepwise program of 200 mM and 250 mM NaCl was applied. This experiment resulted in an excellent separation between BSA monomer (with a calculated mass of 62.4 ± 0.4 kDa), dimer (with a calculated mass of 130 ± 10 kDa), and trimer (with a calculated mass of 170 ± 10 kDa) (Figure 1C). All IEX-MALS experiments show that BSA elutes mostly as a monomer with a purity of 80%, in agreement with SEC-MALS results where BSA monomers elute with a purity of 85%12.
Figure 1: Optimization of IEX-MALS experiment for BSA. (A) IEX-MALS experiment of BSA with a gradient program of 75–350 mM NaCl. (B) IEX-MALS experiment of BSA with a program of a 175 mM NaCl step followed by a linear gradient program of 175–500 mM NaCl. (C) IEX-MALS experiment of BSA with a step program of 200 mM and 250 mM NaCl. The chromatograms display the UV at 280 nm (blue), light scattering at a 90° angle (red), and the refractive index (pink) and the conductivity (grey) curves together with the molar mass of each peak calculated by MALS (black). Please click here to view a larger version of this figure.
IEX-MALS is a powerful method for protein separation and characterization that allows the accurate molar mass determination of pure proteins as well as of heterogeneous samples, characterizing native oligomers, nonnative aggregates, covalent and noncovalent complexes, and conjugated proteins. A program consisting of a linear gradient or a series of salt concentration steps can achieve a good separation of the protein populations and allow proper analysis of each individual peak by the MALS. Further optimization by varying different parameters, such as gradient slope (see step 3.4 of the protocol), can be performed if a better resolution is required. IEX-MALS can be a valuable protein quality control assay since it provides an additional, critical level of protein characterization, complementary to other methods such as SEC-MALS.
While SEC-MALS is a standard and common technique for protein molar mass determination, the relatively low resolution of the standard analytical SEC columns may limit accurate molar mass measurements achieved by MALS12. Some examples of the limitations of SEC-MALS include solutions that contain consecutive oligomers, high levels of aggregation that are not fully separated from the monomer peak, and heterogeneous populations with similar molar masses, such as modified proteins.
IEX is a more complex chromatography method to design and carry out than SEC, but the information obtained from an IEX-MALS experiment can complement and sometimes be even more informative than SEC-MALS analysis. IEX-MALS has successfully characterized antibody variants that share the same molar mass, oligomers that are not completely separated on SEC, and short peptides that are difficult to analyze by SEC12,24. Also, macromolecular assemblies that are too large to be separated by SEC, such as full (containing viral DNA) and empty particles of adeno-associated virus (AAV), can be resolved by IEX25 prior to MALS analysis. Compared to SEC, IEX offers more diverse separation capabilities14 and it has the flexibility of adjusting several parameters to increase peak resolution, such as buffer pH, types of salt, types and length of the column, and others. Unlike SEC, there is no volume limitation for sample injection in IEX, and any molecule can be analyzed independent of its size (see Table 1 in Amartely et al.12). This is a great advantage of IEX-MALS, mainly for samples with a low LS intensity, such as very small proteins or diluted proteins with a tendency for aggregation upon concentration. Since analytical IEX columns are more stable and tend to release fewer particles than SEC columns, IEX-MALS requires very short equilibration time, and LS signals are stabilized very fast. This allows the running of individual experiments as described in this protocol and the stopping of the run as required.
Unlike SEC, which usually provides fine results with only one experiment (using a column with the right fractionation range), IEX may require several experiments to achieve optimal resolution by tuning the method parameters. In IEX-MALS experiments that are performed with a salt gradient, the buffer conductivity and, hence, the RI dramatically change during the run, with consequent changes to the RI signal. This requires an additional blank run for each IEX-MALS experiment and an analysis with baseline subtraction (as described in step 5.2 of the protocol) unless the concentration analysis is limited to UV detection (requiring a priori knowledge of the extinction coefficients for each peak). The analysis with baseline subtraction is robust for linear gradients, even though further development of the method is still required for the successful baseline subtraction of salt stepwise programs. The dn/dc values of each peak should be adjusted according to the specific buffer conductivity at the eluted peak (calculations can be found in the literature12). If a protein eluted at an NaCl concentration lower than 200 mM (like in the BSA example), this adjustment is negligible.
The relatively large amount of protein used in IEX-MALS (as detailed in step 2.3 of the protocol) compared to SEC-MALS is important to overcome the dramatic change of the RI signal caused by the salt gradient and the RI fluctuations due to imperfect mixing of the gradient buffers. If only UV detection is used for mass measurement, smaller amounts may be used. The quantity of protein to inject depends on the molar mass of the protein, homogeneity, purity, and the UV extinction coefficient. The necessary injected mass should be higher for smaller proteins and lower for larger proteins (~1 mg for a 20 kDa protein and ~0.2 mg for a 150 kDa protein). Heterogeneous samples require injection of more sample since the quantity is divided between several populations. Analytical high performance liquid chromatography (HPLC) may require less material than an FPLC system.
Recently, in-line analysis using MALS during purification procedures has been reported. Such a real-time analysis is very efficient for detecting aggregation products that occur during purification and can eliminate the need for any further analysis of the protein after purification26. IEX chromatography is frequently used as an intermediate purification step; thus, the combination of preparative IEX columns with MALS can be useful not only as an analytical characterization method but also as a real-time analysis of large-scale purification procedures. Nonanalytical IEX columns are also stable, with a low degree of particle releasing and, therefore, can be used with MALS. Other separation techniques, such as affinity chromatography or hydrophobic exchange chromatography, can also be combined with MALS when subjected to relatively pure samples (to avoid contamination of the MALS and RI detectors). This will require the adaptation and optimization of the method to obtain not only good peak separation but also sufficiently clean LS and RI signals for a successful MALS analysis.
The authors have nothing to disclose.
The authors thank Dr. Tsafi Danieli (Wolfson Centre for Applied Structural Biology, Hebrew University) for her advice and collaborations. The authors also thank Danyel Biotech Ltd. (Rehovot, Israel) for the assistance and establishment of the analytical FPLC-MALS system utilized in this study.
ÄKTA pure | GE Healthcare | 29-0182-26 | FPLC |
0.02 µm Anotop Whatman Filter 10 mm | GE Healthcare | 6809-1002 | Sample Filter |
0.1 µm Anotop Whatman Filter 10 mm | GE Healthcare | 6809-1012 | Sample Filter |
0.1 µm Anotop Whatman Filter 25 mm | GE Healthcare | 6809-2012 | Mobile phase filter |
BSA (purity >97%) | Sigma | A1900 | Bovine serum albumin |
miniDAWN TREOS | Wyatt Technology | WTREOS | MALS |
mono Q HR 5/50 GL | GE Healthcare | 17-5166-01 | Anion exchange analytical column |
Optilab T-rEX | Wyatt Technology | WTREX | Refractometer |
0.1 mm PES 1000 mL Stericup | Millipore | SCVPU11RE | Mobile phase filter |
Sodium chloride | Sigma | 71382 | HPLC grade NaCl |
TRISMA base | Sigma | T-1503 | TRIS buffer |