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.
For
conjugated HIV-1 fusion peptide vaccine development, recombinant
Tetanus toxoid heavy chain fragment C (rTTHC) was applied as a carrier
protein to boost peptide immunogenicity. Understanding the characteristics
of rTTHC is the first step prior to the peptide conjugation. A comprehensive
mass spectrometry (MS) characterization was performed on E.
coli expressed rTTHC during its purification process. Intact
mass along with peptide mapping analysis discovered the existence
of three cysteine modification forms: glutathionylation, trisulfide
bond modification, and disulfide bond shuffling, in correlation to
a three-peak profile during a hydrophobic interaction chromatography
(HIC) purification step. Coexistence of these multiple oxidative forms
indicated that the active thiols underwent redox reaction in the rTTHC
material. Identity confirmation of the rTTHC carrier protein by MS
analysis provided pivotal guidance to assess the purification step
and helped ensure that vaccine development could proceed.
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