Fab separations for Fab antibodies predicted by QSAR?

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  • Published: Mar 1, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: Laboratory Informatics / Chemometrics & Informatics
thumbnail image: Fab separations for Fab antibodies predicted by QSAR?

Plethora of protein separation methods makes computational techniques attractive

In recent years, many more separation methods have been developed for proteins. In addition to the usual techniques of ion-exchange chromatography (IEC or IEX), size exclusion chromatography (SEC) and hydrophobic interaction chromatography (HIC), a large number of ‘multimodal’ systems, using a wide variety of different ligands have been developed. The sheer number of possible systems presents a problem for scientists wishing to separate proteins, since it may take too long to examine every possibility experimentally, yet simply testing a few systems risks missing the best separation. There has therefore been growing interest in the use of computational methods to predict likely separations.

Quantitative structure–activity relationship (QSAR) calculations have long been a mainstay of medicinal chemistry, being used to determine the likelihood of a particular chemical structure binding to a protein. QSAR calculations have now started to be used to address separation issues, such as predicting the likely separations of proteins by different chromatographic systems.

The Rensselaer/Novo Nordisk researchers investigated the use of QSAR to predict the separations of antibody Fab (Fragment antigen binding) fragments in six separation systems: four multimodal systems, one IEC system (cationic ion exchange, or CEX) and one HIC system.

Fab libraries used in QSAR chromatographic studies

The researchers used Fab libraries made up of structures similar to four wild type Fab species. Suitable descriptors for the antibody fragments were devised, and then used in QSAR computations. A small set of descriptors (four to six) that gave good correlations with the desired response were selected by feature selection calculations. Experimental data were taken from a training set of compounds used in the appropriate chromatographic systems, and a suitable mathematical model was calculated for each system.

A deamidated FAB variant (Fab A LC N33D) was produced and used to test the accuracy of the QSAR predictions on four of the separation columns, all of which were eluted by changing the concentrations of suitable salts (sodium chloride or ammonium sulphate). The experimental retention was significantly less than the predicted retention for two out of the four systems, while being more or less equal for the other two. The order of retention of the Fab A LC N33D on the four columns was correctly predicted (i.e. which column would give the faster elution, which the slowest, etc.).

In another test, three isotype variants (i.e. similar fragments with slight differences in the chains) of one of the wild type Fabs were used in the same four separation systems. For two out of the four separation systems, the differences between the predicted and experimental values for all three variants were less than 0.1 M on a scale of changing salt concentration (all experimental elutions were between ca. 0.05 to ca. 0.55 M salt). Results for the other two systems were less accurate. However, in three out of the four chromatographic systems, the calculations correctly predicted that the three variants would more or less co-elute.

QSAR method for antigen fragments needs further improvement

The QSAR method for antigen fragments gave some correct predictions, but other forecasts were less accurate. Further work is needed to optimise the QSAR procedures. However, there is no doubt that such computational methods will play an increasingly important role in the selection of separation methods.

Related Links

Biotechnology and Bioengineering, 2017, Early View paper. Robinson et al. QSAR models for prediction of chromatographic behavior of homologous Fab variants.

Journal of Chromatography A, 2015, 1407, 58-68. Woo et al. Defining the property space for chromatographic ligands from a homologous series of mixed-mode ligands.

Wikipedia, Fragment antigen-binding

Wikipedia, Quantitative structure–activity relationship

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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