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Abstract: This report describes a novel use of the four-component Ugi reaction to generate a solid-phase library suitable for the purification of immunoglobulins and their fragments by affinity chromatography. An aldehyde-functionalised Sepharose™ solid-support constituted one component in the four-component reaction, whereas the other three components (a carboxylic acid, a primary or secondary amine and an isonitrile) were varied in a combinatorial fashion to generate a tri-substituted peptoidal scaffold structure which provides a degree of rigidity and functionality suitable for rational investigation of immunoglobulin binding. The Ugi ligand library was initially screened chromatographically against whole human IgG and its fragments (Fc and Fab) to yield a Fab-specific lead ligand based on its ability to bind Fab differentially over Fc. Preparative chromatography of IgG from human serum showed 100% of IgG was adsorbed from the 20mg/ml crude stock and subsequently eluted with a purity of 81.0% as determined by SDS-PAGE analysis under non-optimised conditions. High purity Fab and IgG isolation was achieved from both yeast and E. coli host cell proteins according to silver-stained SDS-PAGE lane densitometry. The ligand density and spacer-arm chemistry of the immobilised ligand was optimised to define an affinity adsorbent which binds 73.06mgIgG/ml moist gel (dynamic binding capacity at 10% breakthrough) and a static binding capacity of 16.1±0.25mg Fab/ml moist resin displaying an affinity constant K d =(2.6±0.3)×10−6 M. The lead candidate was modelled in silico and docked into a human Fab fragment (PDB: 1AQK) to suggest a putative binding interface to the constant CH1-CL Fab terminal through six defined hydrogen bond interactions together with putative hydrophobic interactions. [Copyright &y& Elsevier]

Abstract: We have carried out a rigorous evaluation of eight commercially available packed bed chromatography adsorbents for direct capture and purification of immunoglobulins from clarified rabbit antiserum. Three of these materials featured rProtein A (rProtein A Sepharose Fast Flow, Mabselect, Prosep rProtein A) as the affinity ligand, and differed from one another primarily with respect to the underlying base matrix. The remaining five matrices comprised various synthetic low molecular weight ligands immobilised on hydrophilic porous supports and these included: MEP HyperCel, MabSorbent A1P, MabSorbent A2P, FastMabsA and Kaptiv-GY. The general experimental approach taken was to sequentially challenge packed beds of each matrix with a series of different strengths of a clarified antiserum; beginning with the weakest and ending with the strongest. Marked differences in performance (principally evaluated on the basis of dynamic binding capacity, recovery, and purity) were obtained, which allowed clear recommendations concerning the choice of adsorbents best suited for antibody capture from rabbit antisera, to be made. [Copyright &y& Elsevier]

Abstract: A simple mathematical model to predict initial breakthrough profiles from preparative chromatographic separations of biological macromolecules has been developed. A lumped parameter approach was applied, employing Langmuirian adsorption kinetics to describe the rate of mass transfer (MT) from the bulk liquid in the column to the bound state. Equilibrium and kinetic adsorption data were determined for six different packed bed chromatographic adsorbents: two derivatised with rProtein A; and four functionalised with synthetic low molecular weight ligands. All adsorption isotherms were well described by the Langmuir model, whereas the data fitting to kinetic batch experiments showed that the model was inadequate after the first ∼25min of adsorption for four of the six adsorbents. The model underestimated the dynamic Ig breakthough on packed beds of rProtein A Sepharose FF, MabSelect, MBI HyperCel, and MabSorbent A1P, applying a feedstock of 20–100% (v/v) clarified rabbit antiserum. However, when employing a maximum adsorption capacity 25% greater than that determined in batch binding studies, excellent agreement was obtained at all antiserum strengths for most adsorbents. Useful insights into scale-up and process design can be obtained by applying the model, without determining tentative parameters specific for each adsorbent and target protein concentration. However, the model parameters are solvent dependent so a prerequisite for its true applicability is that binding is both Langmuirian and essentially independent of the ionic strength of the feedstock applied. [Copyright &y& Elsevier]

Monoclonal antibodies are key molecules in medicine and pharmaceuticals. A potentially crucial drawback for faster advances in research here is their high price due to the extremely expensive antibody purification process, particularly the affinity capture step. Affinity chromatography materials have to demonstrate the high binding capacity and recovery efficiency as well as superior chemical and mechanical stability. Low-cost materials and robust, faster processes would reduce costs and enhance industrial immunoglobulin purification. Therefore, exploring the use of alternative materials is necessary. In this context, we conduct the first comparison of the performance of magnetic nanoparticles with commercially available chromatography resins and magnetic microparticles with regard to immobilizing Protein G ligands and recovering immunoglobulin G (IgG). Simultaneously, we demonstrate the suitability of bare as well as silica-coated and epoxy-functionalized magnetite nanoparticles for this purpose. All materials applied have a similar specific surface area but differ in the nature of their matrix and surface accessibility. The nanoparticles are present as micrometer agglomerates in solution. The highest Protein G density can be observed on the nanoparticles. IgG adsorbs as a multilayer on all materials investigated. However, the recovery of IgG after washing indicates a remaining monolayer, which points to the specificity of the IgG binding to the immobilized Protein G. One important finding is the impact of the ligand-binding stoichiometry (Protein G surface coverage) on IgG recovery, reusability, and the ability to withstand long-term sanitization. Differences in the materials' performances are attributed to mass transfer limitations and steric hindrance. These results demonstrate that nanoparticles represent a promising material for the economical and efficient immobilization of proteins and the affinity purification of antibodies, promoting innovation in downstream processing.

• Porous alumina monoliths functionalized by single stage silanization with APTES. • Functionalization was found to be extensive and homogeneous. • Temperature and pH were found to have major influence on functionalization efficiency. • Amine functionalized monoliths allows a stable binding of protein A. • Adsorption-elution studies suggested effective use of monoliths for IG purification. In the present work, a simpler and expedited functionalization method was used envisaging the preparation of functionalized alumina monoliths for effective purification of immunoglobulins. A single stage sol-gel synthesis method was used for the silanization of alumina monoliths with (3-Aminopropyl)triethoxysilane (APTES) in an aqueous environment. The nature of the attachment of APTES to the alumina and its distribution through the monolithic column was evaluated through FTIR-ATR, SEM-EDS and XPS measurements. Monolith silanization process was optimized by adjusting the reaction conditions in terms of catalyst used (acid or base), temperature and using a factorial design approach to elicit the interdependent influence of humidity, number of APTES coating layers and precursor concentration on the silanization of alumina. The reaction was found to be optimum at basic pH and a temperature of 80 °C. Etching-free functionalized monoliths with highest amine density of 166 µmol/g of the column were obtained using a single coating with a 2 M APTES solution, at 100% humidity. The binding capacity of the functionalized alumina monolith was determined using BSA and Protein A and then with the immunoglobulin G (IgG) aiming to infer about the suitability of the functionalized alumina monolith for purification of immunoglobulins. Adsorption-elution experiments showed that the functionalized alumina monoliths allowed the attachment of 0.78 mg IgG per mg of bound Protein A and the recovery of 89% of the captured IgG. [ABSTRACT FROM AUTHOR]