“Look closely at what you do early on in process development, because you’ll have to live with those choices at the late stage,” say W. Blaine Stine, Ph.D., associate research investigator at Abbott Laboratories.
Frequently, teams working at the bench seek to optimize activity and minimize toxicity, while being oblivious to physicochemical parameters such as protein solubility and stability. So, Dr. Stine laid out a case study detailing how to manage these features at an early stage during the process of clonal selection after the introduction of back mutations, so as to obtain the overall optimal outcome. This investigation comprised nine IgG antibody-producing clones that had been modified by the introduction of select back mutations into the framework regions of both the light and heavy chains.
Dr. Stine’s tools for characterization include capillary isoelectric focusing, capillary zonal electrophoresis, size exclusion chromatography and differential scanning calorimetry.
The group determined that all nine clones have similar charge isoform distribution, and like-for-like amino acid substitutions did not affect the isoform distributions, while neutral for-charged substitutions resulted in predictable shifts in the entire isoform population. This was apparent for substitutions as small as a single charged amino acid in a protein comprised of over 1,400 residues.
Dr. Stine’s group performed size exclusion chromatography studies to measure the propensity of the molecules to aggregate and fragment and differential scanning calorimetry studies to measure the thermodynamic stability on the nine target clones. The rank order stability determined using the two approaches was shown to be in agreement. Correlating the stability data from the nine clones with the corresponding changes to their amino acid sequence revealed that substitutions at three specific positions appear to affect thermodynamic and accelerated stability.
To better understand this relationship at a mechanistic level, they turned their attention to molecular modeling for the theoretical vindication of their experimental results. The computer modeling results identified three locations where substitutions affected stability. Two of these locations involve a pair of amino acids with closely interacting side chains. The methionine-valine pair was more stable than the isoleucine-alanine pair, which is consistent with the entropic gain from the increase in the number of rotational bonds. The third location was shown to closely interact with an aspartic acid residue.
Experimental data demonstrated that threonine was more stable than valine at that location. This difference is consistent with a molecular model suggesting an unfavorable polar/non-polar interaction with valine present, whereas with threonine there is the potential for the formation of a stabilizing hydrogen bond with the aspartic acid side chain.
These examples support Dr. Stine’s contention that the modeling allows one to develop a mechanistic understanding of how energetically favored substitutions relate with observed changes in physicochemical stability. Moreover, they have the very practical consequence of allowing the investigator to predict the changes that could stabilize a clone which may have favorable binding properties, but would otherwise be excluded because of its molecular fragility.