Overcoming the Multiple-Monomeric-Peak Profile of Broadly Neutralizing HIV-1 Antibody 10E8 with a Unique Size-Exclusion-Chromatography Method.

10E8 is a potent broadly neutralizing antibody (bNAb) that targets the membrane-proximal external region (MPER) of the HIV virus. During early analytical development of this bNAb directed towards clinical evaluation, 10E8 exhibited a multiple-monomeric-peak profile caused by secondary interactions in traditional size-exclusion chromatography (SEC), thereby rendering SEC unfit for the purpose of assessing aggregation, a target critical quality attribute. To overcome this challenge, an innovative and robust SEC method was successfully developed in which the mobile phase was tested for excipients capable of reducing the secondary interactions responsible for the multipeak profile, and an optimal mobile phase composed of 2× PBS and 100 mM arginine at pH 10.55 was established. Application of this optimized mobile phase was shown to allow quantification of the intrinsic level of aggregation of 10E8 without alteration to the SEC matrix itself. Furthermore, the newly developed method was linear, specific, accurate, and precise over an established range. Overall, an SEC method involving optimization of the mobile phase has been successfully developed, which allowed for assessment of antibody aggregation throughout process development, manufacturing, release, and stability testing.

Structures of HIV-1 Neutralizing Antibody 10E8 Delineate the Mechanistic Basis of Its Multi-Peak Behavior on Size-Exclusion Chromatography.

Antibody 10E8 is capable of effectively neutralizing HIV through its recognition of the membrane-proximal external region (MPER), and a suitably optimized version of 10E8 might have utility in HIV therapy and prophylaxis. However, 10E8 displays a three-peak profile on size-exclusion chromatography (SEC), complicating its manufacture. Here we show cis-trans conformational isomerization of the Tyr-Pro-Pro (YPP) motif in the heavy chain 3rd complementarity-determining region (CDR H3) of antibody 10E8 to be the mechanistic basis of its multipeak behavior. We observed 10E8 to undergo slow conformational isomerization and delineate a mechanistic explanation for effective comodifiers that were able to resolve its SEC heterogeneity and to allow an evaluation of the critical quality attribute of aggregation. We determined crystal structures of single and double alanine mutants of a key di-proline motif and of a light chain variant, revealing alternative conformations of the CDR H3. We also replicated both multi-peak and delayed SEC behavior with MPER-antibodies 4E10 and VRC42, by introducing a Tyr-Pro (YP) motif into their CDR H3s. Our results show how a conformationally dynamic CDR H3 can provide the requisite structural plasticity needed for a highly hydrophobic paratope to recognize its membrane-proximal epitope.