Results and Discussion
Weakening the binding affinity between captured antibodies and the Brij
O20 detergent micelle conjugates was investigated at pH 4 and at pH 6.3
by including amino acid monomers during conjugate formation.
(Figure 1) Five amino acid monomers, three with aromatic side
chains and two hydrophobic, were studied: phenylalanine (Phe), tyrosine
(Tyr), tryptophan (Trp), isoleucine (Ile), valine (Val) (Figure
2, A ). Their relative contribution to process efficiency was evidentvia a spiking experiment. Polyclonal, commercial hIgG
(>95% purity) (Figure 2 A, lane 1) was mixed withE. coli lysate, the latter serving as an artificial contaminating
background (Figure 2, A - lane 3 ). The mixture was then added
to the conjugated detergent micelles +/- amino acid monomers. Exclusion
of hydrophilic impurities and extraction of hIgG from the conjugated
micelles at pH 4 (Figure 2, A - lanes 4-9 ) followed. Relatively
pure antibody was obtained with all amino acid monomers tested(Figure 2, B). These findings demonstrated that addition of
amino acid monomers during micelle conjugation leads to improved process
yields when extraction is carried out at pH 4 (Figure 2, B ).
Highest overall yield was obtained when the detergent matrix had been
supplemented with Phe (97%) whereas Ile was the least efficient (85%)(Figure 2, B ).
Increasing the pH to 6.3 during IgG extraction (Figure 2, C ),
produced an average recovery yield of only 54%. As anticipated, we must
conclude that the less acidic conditions are unable to sufficiently
weaken the binding affinity between the bound IgG and the surrounding
detergent matrix. However, when either Phe or Tyr were added during the
conjugation step, overall yields increased dramatically, reaching
82-84%. Trp was found to be the least efficient by
~10% when compared to Phe or Tyr; Ile and Val showed
significant contribution to process yield (76-78%) as compared to yield
in their absence (54%) (Figure 2, C ). Finally, DLS analysis
was also utilized to assess whether recovered hIgG’s extracted at pH 6.3
are monomeric (Figure 3 ). We found that extracted hIgG’s are
indeed monomeric and this was readily observed regardless of the
particular amino acid added to the
Brij-O20:[(bathophenanthroline)3:Fe2+]
conjugates.
In order to begin to rationalize the experimental results described
above, one should distinguish between (i) the downstream effect of
acidic pH on the conformational state of IgG molecules; and (ii) the
nature of the amino acid monomer /nonionic detergent micelle
interaction. The latter may be the basis of our ability to extract
hIgG’s at close to neutral pH and at relatively high purity and yield.
Low pH, during antibody elution from the Protein A column, as well as
during extraction from our conjugated micelle matrix, is liable to
produce a denaturation/renaturation equilibrium, thereby weakening IgG
binding affinity and facilitating extraction on the one hand, but
leading to aggregate formation on the other. Sjogren et al.,[29] have increased our insight into
preferential interaction between peptides and nonionic surfactant
micelles. Their observation of broad NMR signals from tyrosine and
phenylalanine, as compared to lysine, implied that there is a higher
degree of solvent exposure for lysine residues than for the aromatic
side chains, suggesting that peptide and detergent primarily interact
through the aromatic rings of the peptide. Furthermore, the NOESY
spectra displayed NOE cross-peaks between the aromatic ring protons of
the phenylalanine residues and the protons in the surfactant hydrocarbon
chains. This finding confirmed that the aromatic rings and the alkyl
chains of the surfactant are, on average, near each other. All the NMR
results support the conclusion that interactions taking place between
aromatic rings of the peptides and the surfactant alkyl chains are
dominant. We therefore tentatively suggest that the presence of aromatic
rings, e.g. , of phenylalanine or tyrosine, in the hydrophobic
core of the Brij micelles is responsible for weakening the binding of
polyclonal hIgG molecules even at pH 6.3, while not giving rise to
protein denaturation nor to aggregate formation.