Aggregation kills. Aggregated therapeutic proteins can provoke immune response and have reduced therapeutic effect. This has led to regulatory and industrial interest in identifying aggregates for elimination.
Size-exclusion chromatography (SEC/GPC) has been a useful technique for the determination of molecular weights of proteins. However, its usefulness is limited by the requirement for calibration of the SEC/GPC columns with standards comprising proteins of known molecular weight but which may not be of the same chemistry as the unknown protein. Then depending on the eluent it is possible that certain proteins might adhere to the stationary phase during elution, yielding erroneous molecular weight determinations. A better approach is to equip the SEC/GPCsystem with a light scattering detector, enabling accurate molecular weight determinations without the need for column calibration. In this application note, we illustrate the power of this method by confirming the presence of monomeric and dimeric species of bovine serum albumin (BSA) under standard chromatography conditions.
Light scattering data is typically analyzed with the Zimm equation:
Here, K is the Debye constant, a constant of the polymer/solvent system and proportional to the square of the refractive index increment, dn/dc1. Polymer concentration, c, is determined when sample solutions are prepared, and ?R is proportional to the excess scattered intensity and measured by the BI-MwA. q is the scattering vector2.
The ParSEC software calculates absolute concentration and molecular weight as a function of elution volume by generating partial Zimm plots based on the Zimm equation and extrapolating the light scattering data to zero angle. Naturally, extrapolation to zero concentration is impossible since concentration dependent data is unavailable. But, like all modern SEC-LS software ParSEC allows a correction for the second virial coefficient. For the monomer peak, a molecular weight of 67,410 g/mol was obtained, which is in excellent agreement with the expected value of 66,400 g/mol. For the dimer peak, a molecular weight of 130,500 g/mol was obtained which is in excellent agreement with the expected value of 132,800 g/mol.
These data show that the BI-MwA can be used to accurately calculate the absolute molecular weight of monomer and dimer peaks of proteins without the need for column calibration and to quantify the purity of a protein during method development.
|Analyte:||Pierce Biosciences BSA photometric standard|
|Injection Volume:||50 microliters|
|Eluent:||phosphate buffered saline (PBS) at pH 7.5|
|Flow Rate:||0.5 mL/min|
|Column:||TSK-Gel G3000SWXL column|
|UV Wavelength:||280 nm|
|Light Scattering Detector:||BI-MwA|
|Software:||ParSEC “Enhanced” multi-detector chromatography software|