Bertrand Garbay

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.

Cation exchange versus multimodal cation exchange resins for antibody capture from CHO supernatants: Identification of contaminating Host Cell Proteins by mass spectrometry

We compared classical and multimodal cation exchange resins for the capture of recombinant antibodies from Chinese hamster ovary (CHO) cell culture supernatant. Both Capto S and Capto MMC resins present anionic groups while the multimodal Capto MMC also features a hydrophobic moiety. First we screened optimal binding and elution conditions in microplates with a pure antibody. We validated the results on the lab-scale with columns with a pure antibody and a CHO cell culture supernatant. Both resins achieved good yield and purity for the capture step of an antibody. However, the multimodal resin appeared more efficient and selective. Then we identified proteins in the antibody fraction by mass spectrometry in order to highlight the behavior of host cell proteins (HCPs).

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.

Importance of the disulfide bridges in the antibacterial activity of human hepcidin

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. The native peptide of 25 amino acids (Hepc25) contains four disulfide bridges that maintain a β-hairpin motif. The aim of the present study was to assess whether the intramolecular disulfide bridges are necessary for Hepc25 antimicrobial activity. We show that a synthetic peptide corresponding to human Hepc25, and which contains the four disulfide bridges, has an antibacterial activity against several strains of Gram-positive and Gram-negative bacteria. On the contrary, a synthetic peptide where all cysteines were replaced by alanines (Hepc25-Ala) had no detectable activity against the same strains of bacteria. In a further step, the mode of action of Hepc25 on Escherichia coli was studied. SYTOX Green uptake was used to assess bacterial membrane integrity. No permeabilization of the membrane was observed with Hepc25, indicating that this peptide does not kill bacteria by destroying their membranes. Gel retardation assay showed that the Hepc25 binds to DNA with high efficiency, and that this binding ability is dependent on the presence of the intramolecular disulfide bridges. Reduction of Hepc25 or replacement of the eight cysteines by alanine residues led to peptides that were no longer able to bind DNA in the in vitro assay. Altogether, these results demonstrate that Hepc25 should adopt a three-dimensional structure stabilized by the intramolecular disulfide bridges in order to have antibacterial activity.

Correlation between the morphology and the lipid and protein compositions in the peripheral nervous system of individual 8-day-old normal and trembler mice

The hereditary, hypertrophic interstitial neuropathy which afflicts the trembler mouse manifests itself about two weeks after birth. Consequently, the identification of these mutant mice was not possible before this age, except when double mutants were available. We show that the trembler mice can be easily distinguished from their normal littermates before the clinical symptoms appear by using an HPTLC/densitometry technique that allows the simple and rapid analysis of the polar lipids extracted from one sciatic nerve. The results presented in this paper demonstrate important differences between the polar lipid compositions of sciatic nerves from 8-day-old normal and trembler littermates, whose phenotypes were confirmed by the morphological analysis of the contralateral sciatic nerves. The small amount of material that is needed for this identification makes it possible to use the remaining nerve material for other studies. Furthermore, important differences between the sciatic nerve protein compositions of normal and trembler mice, identified according to their polar lipid composition, were also observed and these differences can, therefore, also be employed for the identification of the mutants before the manifestation of the clinical symptoms of the trembler neuropathy.

Expression of the ceramide galactosyltransferase gene during myelination of the mouse nervous system. Comparison with the genes encoding myelin basic proteins, choline kinase and CTP:phosphocholine cytidylyltransferase.

The present study documents the patterns of mRNA expression for the ceramide galactosyltransferase (CGT), the CTP:phosphocholine cytidylyltransferase (CT), and the choline kinase (CK) during the myelination period of the mouse central nervous system (CNS) and peripheral nervous system (PNS). Using the Northern blot technique with densitometric analyses, we show that the CK gene is not developmentally regulated during the period studied, whereas a peak of expression of the CT gene is observed around day 10. On the other hand, the expression of the CGT gene is similar to that of the MBP gene in the CNS and the PNS. Therefore, the synthesis of the galactosylceramides during the myelination period seems to be controlled at the level of the expression of the CGT gene. These results were compared to those of a neurological mutant, the trembler mouse, whose PNS myelination is deficient. Our results clearly indicate that the deficit in the accumulation of the galactosylceramides documented for this mutant is well correlated to a reduced CGT gene expression.

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.

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.

Evidence for the involvement of the anthranilate degradation pathway in Pseudomonas aeruginosa biofilm formation

Bacterial biofilms are complex cell communities found attached to surfaces and surrounded by an extracellular matrix composed of exopolysaccharides, DNA, and proteins. We investigated the whole‐genome expression profile of Pseudomonas aeruginosa sessile cells (SCs) present in biofilms developed on a glass wool substratum. The transcriptome and proteome of SCs were compared with those of planktonic cell cultures. Principal component analysis revealed a biofilm‐specific gene expression profile. Our study highlighted the overexpression of genes controlling the anthranilate degradation pathway in the SCs grown on glass wool for 24 h. In this condition, the metabolic pathway that uses anthranilate for Pseudomonas quinolone signal production was not activated, which suggested that anthranilate was primarily being consumed for energy metabolism. Transposon mutants defective for anthranilate degradation were analyzed in a simple assay of biofilm formation. The phenotypic analyses confirmed that P. aeruginosa biofilm formation partially depended on the activity of the anthranilate degradation pathway. This work points to a new feature concerning anthranilate metabolism in P. aeruginosa SCs.

Po protein in normal, trembler heterozygous/homozygous mice during active PNS myelination

The quantification of P0 protein has been currently used to measure the myelination of the peripheral nervous system (PNS). Using immunological techniques, we show that, as early as postnatal day 8, the P0 content of mice homozygous and heterozygous for the Trembler mutation, represent respectively one tenth and half of the normal values. Thus, although the Trembler mutation is dominant, the myelination defect observed in heterozygous Trembler mice is more pronounced in the homozygous Trembler mice. This gene dosage effect should be taken into account when a molecular model of the Trembler mutation will be proposed.