## 3 Results and Discussion
### Functionalization by Click-Chemistry & Protein Immobilization  
The macroporous materials were chemically modified by click-chemistry to introduce active sites, which are commonly used for protein immobilization on the polymer surface. In a first experiment, propargyl-dPEG1-NHS-ester was attached to the polymer surface which can readily be attacked by nucleophiles present in proteins (e.g., primary amines from lysine). Figure 6 shows the FT-IR spectra of the pristine azide-containing starting material and its characteristic asymmetric azide stretch at 2100 cm\({}^{-1}\). This specific stretch disappears at full conversion, after the 1,3-dipolar cycloaddition yielding a triazole ring. Since the aromatic C-H stretch remains unaffected by the cycloaddition, it can be used as internal reference for semi-quantitative yield determination. In fact, it can be seen that the azide band regressed but did not fully disappear upon the reaction with propargyl-dPEG1-NHS-ester (Figure 6), giving a yield of approximately 55%. Contrarily, the reaction with propargylglycidyl ether gave a yield of 98% (Figure 6).  
After the click reaction, _Staphylococcus aureus_ protein A was immobilized as model protein as it is known to strongly interact with epitopes in the Fc-region of immunoglobulin G antibodies, and therefore relevant for industrial purification of these therapeutic proteins. Hence, protein A was attached to the polymeric scaffold by NHS-amidation or thiol epoxide ring-opening reaction. Two different protein A molecules were used, recombinantly produced protein A with the same primary structure as the native protein and a recombinant version with an introduced cysteine unit at the C-terminus exhibiting a thiol group that can be used for site-selective immobilization. The native-like protein A was linked to the NHS-ester material via multi-point attachment by exploiting the primary amines in protein A's primary structure (1 N-terminus, 27 lysines). Contrarily, the recombinant protein A with the terminal cysteine undergoes a thiol-epoxide ring-opening reaction, which ideally is linked to the polymer via single-point attachment, as illustrated in Figure 7.  
Figure 5: Nitrogen adsorption—desorption cycle giving a TYPE-II isotherm, which is characteristic for macroporous materials. The small hysteresis at higher relative pressures indicates capillary condensation.  
Subsequently, static binding capacities towards monoclonal antibodies were determined. As expected, the single-point attachment using thiol-epoxide linkage revealed a three times higher static binding capacity (\(\sim\)15 mg/mL) towards IgGs compared to the multipoint attachment using the amidation reaction (\(\sim\)5 mg/mL). Since protein A is a globular molecule, most probably the amidation linkage resulted in different orientations, whereas for the thiol-epoxide reaction a uniform orientation can be assumed. Therefore, some protein binding epitopes of protein A are not accessible upon immobilization. The lower capacities can also be attributed to the fact that multipoint attachment leads to alterations in the tertiary structure of protein A and thus to a loss in its biological activity, a phenomenon that has often been reported [15; 36].