| Abstract | Antibody heavy chain variable domains (VHs) form a significant class of biologics. With VH display libraries—the primary source of VH binders—unwanted aggregating VHs are coselected, sometimes overwhelmingly, alongside nonaggregating VHs. Thus, methods enabling efficient screening for nonaggregating VHs are highly valuable. Here, we found that on nonreducing sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) gels, nonaggregating VHs migrate faster than expected, giving underestimated molecular weights (MWs), whereas aggregating ones migrate slower, giving overestimated MWs. Our finding can be applied to large-scale screening for nonaggregating VHs and possibly other proteins, in particular in display library settings, by SDS–PAGE. |
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