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Techniques & Tools Liquid Chromatography

When No Standard is the Gold Standard

Some time ago, we isolated the protein GrpE – a member of the hsp70 protein family. This family of proteins acts as molecular chaperones to support the folding and transport of newly synthesized proteins. To determine molar mass, we applied size-exclusion chromatography (SEC) with conventional calibration, using globulin and albumin standards. The resulting elution volume corresponded with a molecular weight (Mw) of 147 kg/mol.

Were we able to simply sit back and relax? Not a chance! We had in mind a theoretical value of 42 kg/mol and we had nagging doubts about whether there might be something wrong. Did we measure monomers of GrpE? Oligomers? Impurities? Did our standard really behave like the sample and vice versa? Was there an interaction between sample and column? We simply could not get a definite answer with the method we had chosen. So what next? It became clear that an orthogonal method was needed to move forward with our investigation.

Were we able to simply sit back and relax? Not a chance! [...] We had nagging doubts about whether there might be something wrong.

After some consideration, we decided that multi-angle light scattering (MALS) might help solve our problem; after all, light scattering has several interesting features for macromolecular analyses of this type. The main advantage in our case was that MALS has the ability to determine molecular weight without standards – and results are derived only from first principles. In other words, there is a strict correlation used to determine molecular weight:

Mw: Mw = light scattering signal x [analyte].

If the latter term is known – and if it can be measured easily using a UV or RI detector – calculating Mw is relatively straightforward. Consequently, the use of a standard is expendable; data are a result of the calculation instead of conditional comparison.

With MALS, we found a surprisingly different Mw value compared with the data from conventional calibration. Light scattering yielded 41 kg/mol, which is almost exactly what we expected. So, we could claim a successful spot landing!

MALS is even more advantageous when looking at antibodies coupled to conjugates, such as toxins or other effector molecules, for example antibody drug conjugates (ADC). In such cases, ultra-high performance liquid chromatography (UHPLC) SEC columns can improve the separation between closely related compounds significantly. The approach needs less sample, reduces mobile phase consumption and allows shorter run times by using smaller particles in the stationary phase. However, for determining molar mass with a three-detector method (UV, MALS and dRI [differential refractive index]), secondary instrumental band broadening is important, so the narrow peaks obtained from UHPLC require detectors with reduced secondary band broadening.

When it comes to characterization of ADCs, we’ve shown that our three-detector approach allows discrimination between two different preparations and also determination of the drug-antibody ratio (DAR). In other words, for different experimental coupling conditions, it makes it possible to check whether the desired number of drug molecules have bound to the antibody.

In summary, light-scattering technology is able to determine molecular weights of macromolecules without the use of any weight standards because it is an absolute method. Whereas classic calibration failed to generate correct results for various reasons (molecular interaction, adhesion phenomena, shear force distortion), MALS solved our analytical dilemma – and in combination with UV and RI detection, helped give a better insight into molecular composition and the stoichiometry of molecular interactions.

Therefore, in my view, MALS really can serve as a gold standard – using no standards – when it comes to highly complex macromolecular characterization challenges.

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About the Author
Thomas Jocks

Thomas Jocks, Wyatt Technology Europe GmbH, Dernbach, Germany.

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