This protocol presents ion-exchange MALs, a novel method for protein quality control and characterization, quality control that is essential for consistency and integrity of results in life science research and pharmaceutical product development. Ion-exchange MALS determines protein purity, oligomeric state, homogeneity, identity, conformation, structure, post-translation modifications, and other properties. Measurements are non-destructive and are made in solution under near-physiological buffer conditions.

This method accurately determines molar mass of proteins or peptides, the oligomeric state, and the degree of conjugation, for example, glycoproteins. It can also be applied to membrane proteins. IEX separates macromolecules by their surface charge.

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 sample. To begin, use a 0.1-micrometer filter to filter all reagents, including the washing and elution buffers.

Filter the first 50 to 100 milliliters of buffer into a waste bottle in order to eliminate particulates from the dry filters. In a clean, sterile bottle that has been washed thoroughly with filtered water and capped to prevent dust from entering, filter the remainder of the buffer. With pH-eight Tris buffer and 50-millimolar sodium chloride, dilute a BSA sample to six milligrams per milliliter so the pH and ionic strength will allow binding to the ion-exchange column.

Use a syringe to inject at least 0.3 to 0.5 milligrams of BSA into a one-milliliter column for achieving a good-quality MALS analysis. For starting an ion-exchange MALS experiment, open New, Experiment from Method in the MALS software, and select the online method from the Light Scattering system methods folder. If the DLS module is available and DLS data are to be acquired, select the online method from the Light Scattering, With QELS subfolder.

To set the parameters, under the Configuration section, first set the flow rate of the run in the Generic Pump section to the flow rate used in the FPLC as 1.5 milliliters per minute. Under the Solvent section, enter or verify the buffer parameters. Under the Injector section, enter the protein name as BSA, refractive index increment as 0.185 milliliters per gram, and the UV extinction coefficient at the wavelength of 280 nanometers as 0.66 liters per gram per centimeter.

In the Sample tab, enter the concentration of the protein sample as six milligrams per milliliter, and then insert the sample volume for injection as 170 microliters as well. Under the Procedures section, in the Basic Collection tab, select the checkbox Trigger on Auto-Inject, and set the duration of the run to 70 milliliters so that data collection will be continued for at least five minutes after the gradient has reached its final value. Then, in the FPLC software, create a new experiment in the Method Editor tab.

For the initial experiment, create a linear gradient of salt or pH, while for the optimized method create a more specific gradient or a stepwise program according to the manuscript. Include a pulse signal in the method that will trigger data collection in the MALS software. Then, open the pump valve, and wash with 0.5-molar sodium hydroxide to remove strongly bound impurities, followed by a neutralization buffer wash.

Then, wash the column with Tris buffer pH eight, the A valve with washing buffer, and the B valve with elution buffer. Use the washing buffer with the low salt concentration as a final wash to rinse the column, to enable the binding of the protein to the column matrix. Before sample injection, after the wash, and at the end of the elution, the light-scattering baseline must be stabilized to ensure low noise and completely pure dilution.

Using a syringe, place the protein sample in the loop. Start the experiment first in the MALS software by clicking on the Run button and then in the FPLC software. Data will be collected after receiving the pulse signal from the FPLC instrument via the MALS detector.

If the ion-exchanges MALS is performed manually with a continuous flow mode instead of a standalone method, apply the same parameters of the run and instructions as previously described. Once the final method has been verified and run, perform exactly the same method using a blank injection by loading buffer instead of the sample. It is important that the timing between the auto-inject pulse and the gradient of the blank run is identical to that of the sample run.

To begin analysis, under the Procedures section in the MALS software, use the Despiking tab and the Normal level to smooth the chromatograms. If they exhibit a lot of noise, set the level to Heavy. In the Baseline view, define the baseline for all signals, including LS, UV, and RI detectors.

In the Peaks view, define the peaks for analysis. Verify the correct values of RI increment value dn/dc and UV extinction coefficient for the protein under each peak. Under the Molar Mass and Radius from Light Scattering view, analyze the molar mass and the radius using the Zimm model and fit degree of zero.

If QELS is available, under the Rh from QUELS view, observe Rh values and quality of fit of the correlation function. The RI signal changes significantly during the ion-exchange MALS run due to the increase in salt concentration. To subtract the baseline signal from the blank injection, open both the protein and blank ion-exchange MALS experiments.

Right-click on the protein experiment name, select Apply Method, and choose the Baseline Subtraction folder from the file dialog. Select the online method for standard molar mass analysis. Under the Baseline Subtraction view, click Import Blank to import the signals of the blank run.

Under Instruments, check all of the detectors to subtract. To calibrate the ion-exchange MALS system with BSA monomer, under Procedures and Configuration view, align the peaks. Under the Normalization view, enter 3.0 nanometers as the Rg value to perform normalization.

Then, under Band Broadening in the same Configuration tab, choose the peak and use the Perform Fit button to match the UV and LS signals to the RI signal. The graph of the results is shown in the Results Fitting view. Change the axis scales and other graph parameters by right-clicking on the graph, selecting Edit, and then clicking on the Advanced button.

To have more display options, select the EASI Graph tab, and at the top of the window in the Display drop-down menu, select Molar Mass or Rh Q.All results, including molar mass, radius, level of purity, and others are available in the Report view under the Results section. Use the Report Designer button to add more results or parameters, as well as figures, to the report. In this experiment, BSA was analyzed on ion-exchange MALS using an anion exchange analytical column.

The chromatograms displayed the UV at 280 nanometers, light scattering at a 90-degree angle, the refractive index, and the conductivity curves together with the molar mass of each peak. A wide linear gradient consisting of 30 column volumes from 75-millimolar to 350-millimolar sodium chloride separated BSA monomers from dimers and higher oligomers. Downstream MALS analysis resulted in a calculated monomer molar mass of 66.8 kilodaltons and a calculated dimer molar mass of 130 kilodaltons.

Based on the buffer conductivity at the eluted peaks, the gradient was changed to a different program, a long step of 175-millimolar sodium chloride, followed by a linear gradient from 175-millimolar to 500-millimolar sodium chloride. The new gradient greatly improved the resolution and produced excellent separation between BSA monomer and higher oligomeric species. A stepwise program of 200-millimolar and 250-millimolar sodium chloride was applied to focus also on the high oligomeric species.

It resulted in an excellent separation between BSA monomer, dimer, and trimer. Following this procedure, methods such as mass spectrometry, SDS-PAGE activity and stability tests can be performed on each variant separated by ion exchange to identify and further characterize each one. We found that ion-exchange MALS extends the power of MALS analysis to samples that cannot be fully analyzed by SEC-MALS.

Separation by charge resolve distinct species that coelute in SEC.

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