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.