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

Abstract

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.

The influence of proline isomerization on potency and stability of anti-HIV antibody 10E8

“…To investigate the apparent heterogeneity of 10E8 we performed SEC under a series of conditions. As reported previously 7 , 8 , 22 , 10E8 showed elution profiles with multiple peaks. When performing SEC with a monolayer coated silica matrix (Sepax, SRT), three baseline resolved peaks (here denoted as “SEC peaks 1–3”) were observed that eluted much later than expected based on standard protein molecular weight markers (Fig.…”

“…1 B). Previous studies have shown that the SEC resolved 10E8 peaks corresponded to single IgGs that are in a slow dynamic equilibrium 7 , 8 , 22 . To confirm that our 10E8 was similarly behaved we performed SEC with multi-angle light scattering (MALS) and observed three peaks, each of which had a molecular mass near 150 kDa consistent with an intact IgG, as reported previously (Fig.…”

“…Together these data indicate that the Y 100f –P 100g bond isomerization, rather than P 100g –P 100h , govern secondary SEC interactions that have been observed in numerous studies 7 , 9 . Recently it was reported that the SEC peak splitting was alleviated by running in PBS at pH 10.55 and supplementing with 100 mM Arg 8 . The effect of high pH is interesting as the average sidechain pKa of tyrosines in proteins is 10.3 ± 1.2 31 , and thus it may abolish the secondary hydrophobic interaction between the YPP region and the column matrix either directly or by perturbing the kinetics of cis/trans isomerization at the Y–P bond which then alter the SEC secondary interactions.…”

“…While 10E8 can neutralize more than 95% of viral isolates, its poor solubility has presented challenges in its development as a viable therapeutic mAb 6,7 . 10E8 has also been shown to be heterogeneous by size-exclusion chromatography (SEC), a critical assay for development and characterization of potential therapeutic mAbs, for reasons which have yet to be fully elucidated 7,8 . Bispecific antibodies and re-engineered versions of 10E8 have alleviated many of the solubility issues, but still show apparent heterogeneity by SEC 9 .…”

“…Thus far, data implicates the YPP motif in CDRH3 as being the main feature responsible for differences between the SEC resolved 10E8 isomers. We also examined the pepsin-LC-MS profile of 10E8 aggregates, which form under long-term storage and heat-stress 7,8,28 . The aggregated material had a much larger population (61%) of the late eluting C18 peak for peptides at the YPP region (Fig.…”