Aptamers are identified through an iterative process of evolutionary selection starting from a random pool containing billions of sequences. Simultaneously to the amplification of high-affinity candidates, the diversity in the pool is exponentially reduced after several rounds of in vitro selection. Until now, cloning and Sanger sequencing of about 100 sequences was usually used to identify the enriched candidates. However, High-Throughput Sequencing (HTS) is now extensively used to replace such low throughput sequencing approaches. Providing a deeper analysis of the library, HTS is expected to accelerate the identification of aptamers as well as to identify aptamers with higher affinity. It is also expected that it can provide important information on the binding site of the aptamers. Nevertheless, HTS requires handling a large amount of data that is only possible through the development of new in silico methods. Here, this review presents these different strategies that have been recently developed to improve the identification and characterization of aptamers using HTS.

Objective To compare in vitro and in vivo biological and biochemical properties of five liquid intravenous immunoglobulin (IVIg) preparations licensed for therapeutic use in Europe.Methods ClairYg® was compared in a blinded manner to four other liquid IVIg preparations licensed in Europe (Octagam®, Kiovig®, Gamunex®, Privigen®). Three batches of each preparation were tested, except for the IgG repertoires and the animal model.Results Levels of anti-A and anti-B antibodies were lower in ClairYg® (0·11/0·11) relative to a positive EDQM standard and Octagam® (0·11/0·08) than in other preparations (0·33–0·69/0·42–0·46). IgG in ClairYg® recognized 365 and 416 protein spots in HEp-2 cell and Escherichia coli protein extracts vs. 230–330 and 402–842 protein spots, respectively, for IgG in other preparations. IgA content (301 vs. 165–820 ng/mg of IgG), Factor XI and Factor XII antigen (0·46 vs. 0·85–2·40 mU/mg of IgG and 7·8 vs. 20·0–46·2 lU/mg of IgG) C1q binding (0·42 vs. 0·67–1·89 arbitrary units) and C5a uptake (0·41 vs. 0·45–0·66% of activation) were lower in ClairYg® than in other preparations. Finally, intravenous infusion of ClairYg®, Gamunex® and Privigen® had no major effect on arterial blood pressure in spontaneously hypertensive rats.Conclusions Our results evidence some differences in the biological and biochemical properties among licensed liquid IVIg preparations.

Introduction/Aim: LR769 is a new second-generation recombinant human Factor VIIa (rhFVIIa) developed for haemophilia treatment. We determined enzymatic properties of LR769 and its interaction with antithrombin, tissue factor, platelets and endothelial protein C receptor (EPCR), compared with NovoSevenRT. Methods: Kinetic enzyme assays and active site titration were used for enzymatic studies. Surface Plasmon Resonance (SPR) was used for determination of binding constants. Cellular binding was determined for platelets and cultured human umbilical vein endothelial cells (HUVEC). Results: The dissociation constant (K d ) for activated platelet binding was in the 1 lM range for both products. At saturation, more LR769 than NovoSevenRT was bound to the platelets. Binding to HUVEC was 25-50% higher for LR769 than for NovoSevenRT. Protein C, soluble EPCR, and anti-EPCR antibody all reduced the binding, indicating specificity for EPCR. . The K d values for binding of LR769 to soluble tissue factor and full-length tissue factor were 8.1 nM and 0.9 nM, respectively, and the K d for binding to soluble EPCR was 41 nM. Conclusion: Overall, LR769 exhibited characteristics similar to NovoSevenRT, but bound EPCR on HUVEC with somewhat higher affinity than NovoSevenRT.

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Nucleic acid aptamers are promising ligands for analytical and preparative-scale affinity chromatography applications. However, a full industrial exploitation requires that aptamer-grafted chromatography media provide a number of high technical standards that remained largely untested. Ideally, they should exhibit relatively high binding capacity associated to a very high degree of specificity. In addition, they must be highly resistant to harsh cleaning/sanitization conditions, as well as to prolonged and repeated exposure to biological environment. Here, we present practical examples of aptamer affinity chromatography for the purification of three human therapeutic proteins from various sources: Factor VII, Factor H and Factor IX. In a single chromatographic step, three DNA aptamer ligands enabled the efficient purification of their target protein, with an unprecedented degree of selectivity (from 0.5% to 98% of purity in one step). Furthermore, these aptamers demonstrated a high stability under harsh sanitization conditions (100h soaking in 1M NaOH). These results pave the way toward a wider adoption of aptamer-based affinity ligands in the industrial-scale purification of not only plasma-derived proteins but also of any other protein in general.