Many successful, recent therapies for life-threatening diseases such as cancer and rheumatoid arthritis are based on the recognition between native or genetically engineered antibodies and cell-surface receptors. Although naturally produced by the immune system, the need for antibodies with unique specificities and designed for single application, has encouraged the search for novel antibody purification strategies. The availability of these products to the end-consumer is strictly related to manufacture costs, particularly those attributed to downstream processing. Over the last decades, academia and industry have developed different types of interactions and separation techniques for antibody purification, affinity-based strategies being the most common and efficient methodologies. The affinity ligands utilized range from biological to synthetic designed molecules with enhanced resistance and stability. Despite the successes achieved, the purification “paradigm” still moves interests and efforts in the continuous demand for improved separation performances. This review will focus on recent advances and perspectives in antibody purification by affinity interactions using different techniques, with particular emphasis on affinity chromatography.

High blood pressure or hypertension is a condition affecting many individuals and represents a controllable risk factor for cardiovascular diseases such as coronary heart disease and stroke. A non-pharmacological approach to manage these includes the application of food components with antihypertensive activity. Milk protein-derived peptides have been exploited as natural hypotensive agents, namely the peptides {Val-Pro-Pro} {(VPP)} and {Ile-Pro-Pro} {(IPP)}, already commercialized in functional foods as a potential alternative to synthetic drugs. These bioactive peptides inhibit in vitro and in vivo the Angiotensin I-converting enzyme {(ACE)}, a protein with an important role in blood pressure regulation. In this work, we attempted to elucidate the possible mode of interaction between the peptides and {ACE}, including mechanisms of binding to the cofactor Zn2+, and further contrast this with the known mode of inhibition exerted by synthetic drugs {(Captopril}, Enalaprilat and Lisinopril). The bioactive peptide {Ala-Leu-Pro-Met-His-Ile-Arg} {(ALPMHIR)}, also known to inhibit the enzyme {ACE} but with a lower efficiency than {VPP} and {IPP}, was utilized in the docking studies for comparison. It was observed that the best docking poses obtained for {VPP} and {IPP} were located at the {ACE} catalytic site with very high resemblance to the drugs mode of interaction, including the coordination with Zn2+. As for {ALPMHIR}, the best docking poses were located in the narrow {ACE} channel outside the catalytic site, representing higher affinity energies and fewer resemblances with the interaction established by drugs.

Magnetic particles {(MNPs)} offer attractive possibilities in biotechnology. {MNPs} can get close to a target biological entity, as their controllable sizes range from a few nanometres up to tens of nanometres, and their surface can be modified to add affinity and specificity towards desired molecules. Additionally, they can be manipulated by an external magnetic field gradient. In this work, the study of ferric oxide {(Fe3O4)} {MNPs} with different coating agents was conducted, particularly in terms of strategies for antibody attachment at the surfaces (covalent and physical adsorption) and the effects of blocking buffer composition and incubation times on the specific and non-specific interactions observed. The considered biological model system consisted of a coating antibody (goat {IgG)}, bovine serum albumin {(BSA)} as blocking agent, and a complementary antibody labelled with {FITC} (anti-goat {IgG).} The detection of antibody binding was followed by fluorescence microscopy and the intensity of the signals quantified. The ratio between the mean grey values of negative and positive controls, as well as the maximum intensity attainable in positive controls, were considered in the evaluation of the assays efficiency. The covalent immobilization of the coating antibody was more successful as opposed to protein adsorption. For covalent immobilization, silica-coated {MNPs}, a 5% (w/v) concentration of {BSA} in the blocking buffer and incubation times of 1 h produced the best results in terms of assay sensitivity. However, when conducting the assay for incubation periods of 10 min, the fluorescence signal was reduced by 44% but the assay specificity was maintained.