Media Selection in Ion Exchange Chromatography in a Single Microplate.

High-throughput process development is more and more used in chromatography. Limitations are the tools provided by the manufacturers. Here, we describe a method to select ion exchange chromatographic media using a 96-well filter microplate.

Multimodal molecular imaging of atherosclerosis: Nanoparticles functionalized with scFv fragments of an anti-αIIbß3 antibody.

Due to the wealth of actors involved in the development of atherosclerosis, molecular imaging based on the targeting of specific markers would substantiate the diagnosis of life-threatening atheroma plaques. To this end, TEG4 antibody is a promising candidate targeting the activated platelets (integrin αIIbß3) highly represented within the plaque. In this study, scFv antibody fragments were used to functionalize multimodal imaging nanoparticles. This grafting was performed in a regio-selective way to preserve TEG4 activity and the avidity of the nanoparticles was studied with respect to the number of grafted antibodies. Subsequently, taking advantage of the nanoparticle bimodality, both near infrared fluorescence and magnetic resonance imaging of the atheroma plaque were performed in the ApoE-/- mouse model. Here we describe the design of the targeted nanoparticles, and a quantification method for their detection in mice, both ex vivo and in vivo, highlighting their value as a potential diagnosis agent.

Development of anti-chloro 192 tyrosine HDL apoA-I antibodies for the immunodiagnosis of cardiovascular diseases.

High density lipoproteins (HDL) are considered cardio protective. Apolipoprotein A-I (apoA-I), a major component of HDL helps in reverse cholesterol transport, whose function is greatly affected during atherosclerosis due to oxidation by myeloperoxidase. Amino acid tyrosine residue of apoA-I at position 192 and 166 are sensitive to oxidation by myeloperoxidase resulting in the generation of chlorinated and nitrated apoA-I and they are believed to be present in atherosclerotic plaques and in circulation. These oxidized apoA-I have been suggested as potential indicator(s) of CVD risks in humans. To detect the levels of oxidized apoA-I there is a need for developing monoclonal antibodies (mAbs) with high specificity and sensitivity that could be utilized routinely in clinical immune based assays for blood plasma or for in vivo imaging. In this study, chemically chlorinated apoA-I (chlorinated 192tyrosine- apoA-I) and a short synthetic peptide, containing the corresponding chlorinated tyrosine residue, conjugated to keyhole limpet hemocyanin (KLH) carrier protein were used for immunization. Stable hybridoma clones F7D5 and G11E3 were found to be highly sensitive and reactive towards chlorinated 192tyrosine- apoA-I. Interestingly, these mAbs also displayed positive reaction with atherosclerotic plaques obtained from mouse and human biopsies. In vitro or in vivo diagnostic tests could be developed either by detecting oxidized apoA-I in human plasma or by directly imaging atheroma plaques as both mAbs were shown to stain human atheroma. The anti-chlorinated 192tyrosine- apoA-I mAbs described in this study may have a high diagnostic potential in predicting CVD risks.

Mixed Mode Chromatography: A Novel Way Toward New Selectivity.

Mixed mode chromatography offers a diversity of ligands, each providing a new selectivity. This allows the design of novel purification processes with reduced column steps. Structure of ligands is based on both hydrophobic and ionic groups. Thanks to its salt tolerance, crude extracts or post-IEX samples can be loaded directly without conditioning. The selectivity could be enhanced by modulating elution parameters or by using additives. More importantly, mixed mode chromatography could be as effective as affinity chromatography for mAb purification processes. Mixed mode chromatography opens the way to short and economical processes.

Mixed Mode Chromatography, Complex Development for Large Opportunities.

Mixed mode chromatography resins with salt tolerance, large design space and orthogonal selectivity requires a slightly more complex development than traditional resins. It is important to screen several ligands and several binding and elution conditions. This allows taking full advantage of these resins. High-Throughput Screening (HTS) for Process Development should be done with the help of Design of Experiment (DoE). It could be performed in filter plates or Robocolumns, and assisted by liquid handling automated workstation. Modeling of the results allows the choice of optimal parameters that can then be validated and scaled up. All this leads to a better knowledge and robustness of the purification step.

Comparative study of strong cation exchangers: Structure-related chromatographic performances.

Chromatographic performances are highly influenced by operational parameters. New ion exchangers have tailored matrices providing low backpressure, thereby allowing high flow velocity. By systematic frontal analysis and selectivity determination at different flow rates, we independently evaluated cation exchangers to facilitate media selection and investigated the relationship between surface modification and chromatographic performances. Structure-extended resins showed higher binding capacities compared to resins with conventional ligands directly attached to the matrix. Moreover, they maintained high capacities even with high flow velocities. Ligand accessibility was therefore largely enhanced, allowing proteins to interact and bind under harsh conditions with minimal residence/contact time. High throughput resins can be used for purification of high volume and high concentration feedstock in limited time. This results in higher productivity, and could contribute to cost reduction. In this work, we evaluated the dynamic binding capacities of various new ion exchange resins at different binding conductivities for different residence times, and observed that.

Role of Glycanation and Convertase Maturation of Soluble Glypican-3 in Inhibiting Proliferation of Hepatocellular Carcinoma Cells.

Glypican 3 (GPC3) is a complex heparan sulfate proteoglycan associated with the outer surface of the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. It is also N-glycosylated and processed by a furin-like convertase. GPC3 has numerous biological functions. Although GPC3 is undetectable in normal liver tissue, it is abnormally and highly overexpressed in hepatocellular carcinoma (HCC). Interestingly, proliferation of HCC cells such as HepG2 and HuH7 is inhibited when they express a soluble form of GPC3 after lentiviral transduction. To obtain more insight into the role of some of its post-translational modifications, we designed a mutant GPC3, sGPC3m, without its GPI anchor, convertase cleavage site, and glycosaminoglycan chains. The highly pure sGPC3m protein strongly inhibited HuH7 and HepG2 cell proliferation in vitro and induced a significant increase in their cell doubling time. It changed the morphology of HuH7 cells but not that of HepG2. It induced the enlargement of HuH7 cell nuclear area and the restructuration of adherent cell junctions. Unexpectedly, for both cell types, the levels of apoptosis, cell division, and ß-catenin were not altered by sGPC3m, although growth inhibition was very efficient. Overall, our data show that glycanation and convertase maturation are not required for sGPC3m to inhibit HCC cell proliferation.

A Recombinant Human Anti-Platelet scFv Antibody Produced in Pichia pastoris for Atheroma Targeting.

Cells of the innate and adaptive immune system are key factors in the progression of atherosclerotic plaque, leading to plaque instability and rupture, potentially resulting in acute atherothrombotic events such as coronary artery disease, cerebrovascular disease and peripheral arterial disease. Here, we describe the cloning, expression, purification, and immunoreactivity assessment of a recombinant single-chain variable fragment (scFv) derived from a human anti-αIIbß3 antibody (HuAb) selected to target atheromatous lesions for the presence of platelets. Indeed, platelets within atheroma plaques have been shown to play a role in inflammation, in platelet-leucocyte aggregates and in thrombi formation and might thus be considered relevant biomarkers of atherosclerotic progression. The DNA sequence that encodes the anti-αIIbß3 TEG4 scFv previously obtained from a phage-display selection on activated platelets, was inserted into the eukaryote vector (pPICZαA) in fusion with a tag sequence encoding 2 cysteines useable for specific probes grafting experiments. The recombinant protein was expressed at high yields in Pichia pastoris (30 mg/L culture). The advantage of P. pastoris as an expression system is the production and secretion of recombinant proteins in the supernatant, ruling out the difficulties encountered when scFv are produced in the cytoplasm of bacteria (low yield, low solubility and reduced affinity). The improved conditions allowed for the recovery of highly purified and biologically active scFv fragments ready to be grafted in a site-directed way to nanoparticles for the imaging of atherosclerotic plaques involving inflammatory processes and thus at high risk of instability.

High yield of recombinant human Apolipoprotein A-I expressed in Pichia pastoris by using mixed-mode chromatography.

A vast majority of the cardioprotective properties exhibited by High-Density Lipoprotein (HDL) is mediated by its major protein component Apolipoprotein A-I (ApoA1). In order to develop a simplified bioprocess for producing recombinant human Apolipoprotein A-I (rhApoA1) in its near-native form, rhApoA1was expressed without the use of an affinity tag in view of its potential therapeutic applications. Expressed in Pichia pastoris at expression levels of 58.2 mg ApoA1 per litre of culture in a reproducible manner, the target protein was purified by mixed-mode chromatography using Capto™ MMC ligand with a purity and recovery of 84% and 68%, respectively. ApoA1 purification was scaled up to Mixed-mode Expanded Bed Adsorption chromatography to establish an 'on-line' process for the efficient capture of rhApoA1 directly from the P. pastoris expression broth. A polishing step using anion exchange chromatography enabled the recovery of ApoA1 up to 96% purity. Purified ApoA1 was identified and verified by RPLC-ESI-Q-TOF mass spectrometry. This two-step process would reduce processing times and therefore costs in comparison to the twelve-step procedure currently used for recovering rhApoA1 from P. pastoris.

Purification process of recombinant monoclonal antibodies with mixed mode chromatography.

An innovative process to purify mAb from CHO cell culture supernatant was developed. This three-step process involved two mixed mode resins and an anion exchange membrane. We used a human IgG mixture to determine the optimal conditions for each purification step. Thereafter, the whole process was evaluated and improved for the purification of a recombinant mAb produced in the supernatant of CHO cells. Once optimized, yield and purity of 88% and 99.9%, respectively were comparable to those obtained in a conventional process based on a capture step using protein A. In addition, aggregates, HCPs and DNA levels in the purified fraction were below regulatory specifications. Then we used mass spectrometry to identify contaminating proteins in the antibody fraction in order to highlight the behavior of HCPs.

Production and purification of recombinant human hepcidin-25 with authentic N and C-termini.

Hepcidin was first identified as an antimicrobial peptide present in human serum and urine. It was later demonstrated that hepcidin is the long-sought hormone that regulates iron homeostasis in mammals. Recombinant human Hepcidin-25 (Hepc25) was expressed in Pichia pastoris using a modified version of the pPICZαA vector. Hepc25 was then purified by a simple two-step chromatographic process to obtain 1.9 mg of soluble recombinant human Hepc25 per liter of culture at 96% purity. The sequence of Hepc25 and the presence of four disulfide bridges were confirmed by mass spectrometry analyses, and the recombinant Hepc25 exhibited antibacterial activity. This protocol of production and purification is the first step toward the production of human Hepc25 at a greater scale.

Media selection in ion-exchange chromatography in a single microplate.

High-throughput process development is more and more used in chromatography. Limitations are the tools provided by the manufacturers. Here, we describe a method to select chromatographic media for ion-exchange chromatography using a 96-well filter microplate.

Electrodeposition of polymer nanodots with controlled density and their reversible functionalization by polyhistidine-tag proteins.

We present a simple and rapid procedure for producing polymer-coated substrates that can be easily functionalized by ion-chelating proteins. The procedure consists of depositing 18 nm metal-chelating cyclam-modified polymer nanoparticles (cyclam-nps) onto a conductive substrate (an Indium Tin Oxide (ITO) electrode) from an aqueous dispersion of Cu(2+)-loaded cyclam-nps while being subjected to a direct current (DC) field. The density of deposited nps as measured by AFM is shown to be in direct correlation to the concentration of nps in the dispersion with deposition of the particles taking less than 5 s. Because of the functionalization of the nps with cyclam groups, they can be used as anchoring sites for 6-Histidine (6-His) tagged proteins through complexation with divalent metal ions. In this work 6-His Green Fluorescent Protein (6-His GFP) is used as a model protein. The characterization by fluorescence microscopy clearly shows that the protein affinity was ion dependent and that the 6-His GFP density can be controlled by np density, which is itself easily tunable. AFM observations confirmed the immobilization of 6-His GFP onto cyclam-nps and its subsequent removal by treatment with ethylenediaminetetraacetic acid (EDTA).

In vivo phage display to identify new human antibody fragments homing to atherosclerotic endothelial and subendothelial tissues [corrected].

In vivo phage display selection is a powerful strategy for directly identifying agents that target the vasculature of normal or diseased tissues in living animals. We describe here a new in vivo biopanning strategy in which a human phage single-chain antibody (scFv) library was injected into high-fat diet-fed ApoE(-/-) mice. Extracellular and internalized phage scFvs were selectively recovered from atherosclerotic vascular endothelium and subjacent tissues. After three successive biopanning rounds, a panel of six clones with distinct gene sequences was isolated. Four scFvs produced and purified in soluble form were shown to interact in vitro with a rabbit atheromatous protein extract by time-resolved fluorescence resonance energy transfer and to target the endothelial cell surface and inflamed intima-related regions of rabbit and human tissue sections ex vivo. These new scFvs selected in a mouse model recognized both rabbit and human tissue, underlying the interspecies similarities of the recognized epitopes. By combining immunoprecipitation and mass spectrometry, one of the selected scFvs was shown to recognize carbonic anhydrase II, an up-regulated enzyme involved in resorption of ectopic calcification. These results show that in vivo biopanning selection in hypercholesterolemic animals makes it possible to identify both scFvs homing to atherosclerotic endothelial and subendothelial tissues, and lesion-associated biomarkers. Such scFvs offer promising opportunities in the field of molecular targeting for the treatment of atherosclerosis.

Antibody capture by mixed-mode chromatography: a comprehensive study from determination of optimal purification conditions to identification of contaminating host cell proteins.

We evaluated mixed mode chromatography for the capture of recombinant antibodies from CHO cell culture supernatants. We studied PPA HyperCel, HEA HyperCel, MEP HyperCel and Capto adhere resins, which all contain hydrophobic and cationic groups. A microplate approach combined with DoE modeling allowed the exploration of the complex behaviors of these mixed mode resins. Optimal conditions for antibody purification and host cell proteins (HCPs) elimination were determined and then directly up-scaled to laboratory columns. Then we used mass spectrometry to identify the major HCPs potentially coeluted with the antibody. Differences between the four resins in terms of amount, complexity and identity of the HCPs present in the elution fractions were investigated.

Evidence for specific interaction between the RhoGAP domain from the yeast Rgd1 protein and phosphoinositides.

The Rho GTPase activating protein Rgd1 increases the GTPase activity of Rho3p and Rho4p, which are involved in bud growth and cytokinesis, respectively, in the budding yeast Saccharomyces cerevisiae. Rgd1p is a member of the F-BAR family conserved in eukaryotes; indeed, in addition to the C-terminal RhoGAP domain Rgd1p possesses an F-BAR domain at its N-terminus. Phosphoinositides discriminate between the GTPase activities of Rho3p and Rho4p through Rgd1p and specifically stimulate the RhoGAP activity of Rgd1p on Rho4p. Determining specific interactions and resolving the structure of Rgd1p should provide insight into the functioning of this family of protein. We report the preparation of highly pure and functional RhoGAP domain of Rgd1 RhoGAP domain using a high yield expression procedure. By gel filtration and circular dichroïsm we provide the first evidences for a specific interaction between a RhoGAP domain (the RhoGAP domain of Rgd1p) and phosphoinositides.

Structure-activity relationship of human liver-expressed antimicrobial peptide 2.

Liver-expressed antimicrobial peptide 2 (LEAP-2) is a 40-residue cationic peptide originally purified from human blood ultrafiltrate. The native peptide contains two disulfide bonds and is unique regarding its primary structure. Its biological role is not known but a previous study showed that chemically synthesized LEAP-2 exhibited in vitro antimicrobial activities against several Gram-positive bacteria. In order to determine its antimicrobial mode of action, we expressed human recombinant LEAP-2 in Escherichia coli. Circular dichroism spectroscopy and nuclear magnetic resonance analyses showed that the structure of the recombinant peptide was identical to that of the chemically synthesized and oxidized LEAP-2, with two disulfide bonds between Cys residues in relative 1-3 and 2-4 positions. Minimal inhibitory concentration (MIC) of the recombinant human LEAP-2 was determined by a conventional broth dilution assay. It was found to be bactericidal against Bacillus megaterium at a 200microM concentration. Interestingly, the linear LEAP-2 had a greater antimicrobial activity with a MIC value of 12.5microM, which was comparable to that of magainin2. SYTOX Green uptake was used to assess bacterial membrane integrity. Linear LEAP-2 and magainin2 permeabilized B. megaterium membranes with the same efficiency, whereas oxidized LEAP-2 did not induce stain uptake. Binding of the peptides to plasmid DNA was evaluated by gel retardation assays. The DNA-binding efficacy of linear LEAP-2 was three times higher than that of the peptide-containing disulfide bridges. Altogether, these results show that the secondary structure of human LEAP-2 has a profound impact on its antibacterial activity.

Comparative study of strong anion exchangers: structure-related chromatographic performances.

Chromatographic performances are highly influenced by operational parameters. New ion exchangers have tailored matrices providing low backpressure and allowing high flow velocity. By systematic frontal analysis and selectivity determination at different flow rates, we suggested an independent evaluation of major anion exchangers to facilitate media selection, and investigated the relationship between (i) surface modification and (ii) chromatographic performances. Structure-extended resins showed higher binding capacities compared to resins with conventional ligands directly attached to the matrix. Moreover, they maintained mainly high capacities even with extremely high flow velocities. Ligand accessibility was therefore largely enhanced, allowing proteins to interact and bind under harsh conditions. High throughput resins can be used for purification of high volume and high concentration feedstock in limited time. This results in higher productivity and could contribute to cost reduction.

Efficient purification of recombinant proteins fused to maltose-binding protein by mixed-mode chromatography.

Two mixed-mode resins were evaluated as an alternative to conventional affinity resins for the purification of recombinant proteins fused to maltose-binding protein (MPB). We purified recombinant MBP, MBP-LacZ and MBP-Leap2 from crude Escherichia coli extracts. Mixed-mode resins allowed the efficient purification of MBP-fused proteins. Indeed, the quantity of purified proteins was significantly higher with mixed-mode resins, and their purity was equivalent to that obtained with affinity resins. By using purified MBP, MBP-LacZ and MBP-Leap2, the dynamic binding capacity of mixed-mode resins was 5-fold higher than that of affinity resins. Moreover, the recovery for the three proteins studied was in the 50-60% range for affinity resins, and in the 80-85% range for mixed-mode resins. Mixed-mode resins thus represent a powerful alternative to the classical amylose or dextrin resins for the purification of recombinant proteins fused to maltose-binding protein.