Immunoglobulin adsorption on modified surfaces

Preservation of biological functioning of proteins during immobilisation is of special interest in various biomedical and biotechnical applications. In industry physical adsorption of immunoglobulins (IgGs) onto solid surfaces is still the predominant immobilisation procedure because it is relatively easy to perform. Physical adsorption, however, often results in an undesired loss of biological activity. This loss of activity may be caused by changes in the specific folding (the conformation) of the IgG or by a reduced accessibility of the antigen binding sites by blocking, for instance when IgG molecules are adsorbed with their antigen binding sites oriented towards the sorbent surface.

To control the orientation and conformation of adsorbed IgG molecules we studied the interactions involved in physical adsorption of IgG molecules on solid surfaces. Our goal was to optimize the biological activity of adsorbing IgG molecules. For this, we introduced a new method to achieve oriented physical adsorption of IgG. This concept is based on the anisodimensionality of IgG molecules and resembles 'molecular sieving' on the sorbent surface. The 'sieve' is created by preadsorbed molecules that form a steric barrier preventing adsorption at some sites, but leaving patches of uncovered surface area. The open areas in the preadsorbed layer can be tuned in such a way that only the smaller part of anisodimensional molecules can enter and adsorb. In the case of 19G this means that only the Fc part can adsorb to the surface and thereby forcing the larger antigen binding parts (F(ab) ₂ ), directed towards the solution and, hence, accessible to bind antigens. A 'sieve' formed either with preadsorbed IgG molecules or with triblock copolymers of poly(ethylene oxide), PEO, and poly(propylene oxide), PPO, of the type PEOPPO-PEO was proven to yield a higher specific biological activity of subsequently adsorbing IgG.

In brief, the effect of a 'molecular sieve' on IgG adsorption is essentially threefold. Firstly, it induces oriented IgG adsorption. Secondly, it prevents extended undesirable structural changes in adsorbed IgG and thirdly, it prevents undesirable reorientation of the adsorbed IgG.

In chapter 1 it is explained that in many applications there is a need to control the biological activity of adsorbed IgG. The physical properties and characteristics of

IgG molecules and the interactions involved in physical adsorption of proteins are described and, more importantly, our variant of 'molecular sieving' is introduced. Finally, an outline of this thesis is given.

The influence of electrostatic interactions on the adsorption of IgG is examined both theoretically and experimentally in chapter 2. The long range interaction between IgG and the sorbent surface is treated in terms of the DLVO theory. We attempted to make use of the dipolar character of the IgG molecules to control their orientation upon adsorption. It is concluded that electrostatic interactions have a strong influence on the adsorption behaviour of IgG molecules on hydrophilic charged surfaces. Due to extensive desorption of IgG from both positively and negatively charged surfaces, electric field-induced orientation of IgG could not be established unambiguously.

Chapter 3 is mainly dedicated to the orientational aspects of IgG adsorption. In this chapter the phenomenon of 'molecular sieving' is demonstrated first theoretically using a Random Sequential Adsorption (RSA) model and second experimentally by a set of reflectometry experiments on surfaces partially covered with preadsorbed layers of either IgG or triblock copolymers of PEO-PPO-PEO. The rate of IgG adsorption and the maximum adsorbed amount decreases with increasing adsorbed amount of triblock copolymer. On the precoated layers, IgG is indeed adsorbed in a preferential orientation which yielded a higher specific biological activity of the IgG molecules. Furthermore, we observed that the preadsorbed layers prevent undesirable reorientation of adsorbed IgG.

The mass flux towards the surface also has a profound effect on the adsorbed amount and, consequently, on the orientation of IgG.

In chapters 4, 5 and 6 conformational changes in IgG are studied. In chapter 4 a structural analysis of a monoclonal lgG adsorbed on different silica surfaces (hydrophilic, hydrophobic, hydrophobic with preadsorbed triblock copolymers) using ATR-FTIR spectroscopy is given. The secondary structure of adsorbed IgG on a hydrophilic silica surface resembles that of native 19G in solution. The presence of preadsorbed triblock copolymers on the hydrophobic silica surface cause a decrease in the adsorbed amount of IgG and, more importantly prevent substantial structural rearrangements in the adsorbed IgG molecules.

In chapters 5 and 6, Circular Dichroism (CD) is used as a spectroscopic technique for studying protein structure in the adsorbed state. Chapter 5 gives information on the structural changes of IgG molecules induced by adsorption on a hydrophobic

surface and compares these changes with those induced by heat treatment. Neither heatinduced nor adsorption-induced structural changes lead to complete unfolding into an extended polypeptide chain, but leave a significant part of the IgG molecule in a globular or corpuscular form. The structural changes induced by heating and by adsorption are different.

The effect of preadsorbed IgG and triblock copolymer molecules on the secondary structure of subsequently adsorbing IgG molecules is studied in chapter 6. Structural rearrangements were less extensive with increasing surface coverage of the polymer. It was found that preadsorbed IgG molecules have comparable effects on the secondary structure of subsequent adsorbing IgG; a more native-like structure is retained for the higher adsorbed amounts. Hence, partial pre-coating of a surface is an effective way to control the secondary structure of later adsorbed IgG molecules.

To examine whether the model results apply to industrially manufactured diagnostic methods we implemented in chapter 7 the triblock copolymer preadsorption procedure in a microplate assay. Radio-actively labelled IgG and hCG molecules allowed us to monitor the adsorption of IgG and the subsequent specific binding of hCG. The data obtained are in agreement with our earlier model studies and demonstrate that sieving of the IgG by the polymer does take place, resulting in the creation of a more favourably oriented IgG layer. Our studies indicate that the amount of precoated material is critical in the formation of an operational 'sieve'.