Factors That Influence Adsorption

The major types of interactions that are relevant in immunoglobulin adsorption from aqueous solution are (1) hydrophobic interaction, (2) Coulomb interaction, and (3) hydrogen bonding. The effects of electrostatic charge and potential (which can be controlled by varying the pH and ionic strength in the system), the hydrophobicities of the protein and the sorbent surface, and the chemical compositions of the sorbent and the medium on the rate of adsorption and on the adsorbed amount at equilibrium provide insight into the relative importance of the above-mentioned interactions. Other factors that may also influence im-munoglobulin adsorption onto surfaces include intermolecular forces between adsorbed molecules, solvent-solvent interactions, strength of functional group bonds, chemistry of solid surface, topology, and morphology. Adsorption of IgG on hydrophobic surfaces is usually an irreversible process and occurs rapidly. It

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Adsorption pH

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Adsorption pH

FIG. 4 Maximum adsorption of monoclonal IgG on cationic (▲) and anionic (•) PS latex as a function of pH at 2 mM ionic strength and (20 ± 1)°C.

should be noted that the characteristics of F(ab')2 and IgG adsorption are very similar [45,46].

The major driving force for protein adsorption onto polymer surfaces is the dehydration of hydrophobic side groups [11,47], which is almost completely due to the entropy increase in water that is released from contact with hydrophobic components, and the surface dehydration also favors the protein adsorption. It seems reasonable to assume that antibody adsorption on hydrophobic surfaces is driven entropically as well. However, electrostatic forces at low ionic strength can play a certain role in IgG adsorption even on hydrophobic surfaces. This role has been shown by the adsorption of monoclonal antibodies onto surfaces with different signs of surface charge [36,38,43,48]. The initial slopes in the adsorption isotherms give information about the affinity between IgG and the adsorbent surface. With this aim, several authors have studied adsorption isotherms on systems that vary the possible electrostatic interactions between the components. Figure 5 shows the adsorption of a monoclonal antibody (IEP 5.5) at neutral adsorption pH on positively and negatively charged surfaces. We can see the differences in affinity between the IgG molecules and the polymer surfaces when the electrostatic forces influence the adsorption process. Effectively, e e

FIG. 5 Adsorption isotherms of monoclonal IgG on cationic (•) and anionic (▲) PS latex at pH 7, 2 mM ionic strength and (20 ± 1)°C.

FIG. 5 Adsorption isotherms of monoclonal IgG on cationic (•) and anionic (▲) PS latex at pH 7, 2 mM ionic strength and (20 ± 1)°C.

the initial adsorption values for the anionic surface do not coincide with the total adsorption line, showing that electrostatic repulsion between negative charges makes the approach of IgG molecules to the surface difficult.

The electrostatic forces can give rise to fractionation in the adsorption of polyclonal antibodies on charged surfaces. Since polyclonal antibodies are in fact mixtures of IgG molecules with different physical properties, preferential adsorption of any fraction can take place at low ionic strength. To check this possibility, some authors [43] have analyzed by isoelectrofocusing (IEF) the supernatants after IgG adsorption onto charged surfaces. These authors have demonstrated that, at pH 7 and 9, preferential adsorption is partly determined by electrostatic factors; the IgG molecules with the highest IEP are preferentially adsorbed on negatively charged surfaces, whereas at pH 5 no preferential adsorption is observed. Most single-component IgG adsorption from buffer studies simulates physiological conditions, implying that the ionic strength is relatively high. Under those experimental conditions the electrostatic forces between protein and adsorbent are negligible. An exception is the serum competition, where IgG adsorption from a multicomponent protein solution is a phenomenon completely distinct from single-component IgG adsorption from buffer.

Antibody adsorption to, and desorption from, adsorbent surfaces is a function of the nature of both antibody and the surface, and can be dependent on time, temperature, ionic strength, pH, protein concentration, and surface tension [39]. IgG molecules adsorbed onto a surface are in a dynamic state. Although adsorbed IgG molecules generally do not desorb as a result of simple dilution, they can be displaced by an increase in ionic strength. Certainly, ionic strength exerts a pronounced effect on the adsorption of IgG molecules on charged surfaces. As ionic strength increases, the electrostatic forces between the IgG molecules and the adsorbent decreases. Under these experimental conditions, hydro-phobic interactions are predominant in the adsorption mechanism of IgG molecules on a surface. Figure 6 shows the plateau values of adsorption as a function of pH at increasing ionic strength. In this case, the adsorbed IgG maximum is less dependent on pH at high ionic strength. Nevertheless, the effect of ionic strength on the adsorbed amount is different at adsorption pH 4, 7, and 10, as can be seen in Fig. 7. At neutral pH an increase in the ionic strength

FIG. 6 Maximum adsorption as a function of pH at different NaCl ionic strengths for rabbit IgG: 2 mM (■), 20 mM (▲), 50 mM (•), 100 mM (▼).

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FIG. 7 Maximum adsorption as a function of NaCl ionic strength at different adsorption pH for rabbit IgG: pH 4 (▲), pH 7 (•), pH 9 (■).

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Ionic Strength (mM)

FIG. 7 Maximum adsorption as a function of NaCl ionic strength at different adsorption pH for rabbit IgG: pH 4 (▲), pH 7 (•), pH 9 (■).

implies a decrease in the plateau value, whereas at pH 4 and 10 this value increases. This trend seems to indicate that the structural stability of polyclonal IgG molecules decreases as ionic strength increases at neutral pH, whereas when the polypeptide chains are highly charged (pH 4 and 10) an increase in electrolyte concentration provokes a larger screening of the net charge on the IgG molecules and, thus, an increasing conformational stability of the IgG.

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