Selective antibody precipitation using polyelectrolytes: a novel approach to the purification of monoclonal antibodies.

We evaluated the potential for polyelectrolyte induced precipitation of antibodies to replace traditional chromatography purification. We investigated the impact of solution pH, solution ionic strength and polyelectrolyte molecular weight on the degree of precipitation using the anionic polyelectrolytes polyvinylsulfonic acid (PVS), polyacrylic acid (PAA), and polystyrenesulfonic acid (PSS). As we approached the pI of the antibody, charge neutralization of the antibody reduced the antibody-polyelectrolyte interaction, reducing antibody precipitation. At a given pH, increasing solution ionic strength prevented the ionic interaction between the polyelectrolyte and the antibody, reducing antibody precipitation. With increasing pH of precipitation, there was an increase in impurity clearance. Increasing polyelectrolyte molecular weight allowed the precipitation to be performed under conditions of higher ionic strength. PVS was selected as the preferred polyelectrolyte based on step yield following resolubilization, purification performance, as well as the nature of the precipitate. We evaluated PVS precipitation as a replacement for the initial capture step, as well as an intermediate polishing step in the purification of a humanized monoclonal antibody. PVS precipitation separated the antibody from host cell impurities such as host cell proteins (HCP) and DNA, process impurities such as leached protein A, insulin and gentamicin, as well as antibody fragments and aggregates. PVS was subsequently removed from antibody pools to < 1 microg/mg using anion exchange chromatography. PVS precipitation did not impact the biological activity of the resolubilized antibody.

Fragments of protein A eluted during protein A affinity chromatography.

Protein A affinity chromatography is a common method for process scale purification of monoclonal antibodies. During protein A affinity chromatography, protein A ligand co-elutes with the antibody (commonly called leaching), which is a potential disadvantage since the leached protein A may need to be cleared for pharmaceutical antibodies. To determine the mechanism of protein A leaching and characterize the leached protein A, we fluorescently labeled the protein A ligand in situ on protein A affinity chromatography media. We found that intact protein A leaches when loading either purified antibody or unpurified antibody in harvested cell culture fluid (HCCF), and that additionally fragments of protein A leach when loading HCCF. The leaching of protein A fragments can be reduced by EDTA, suggesting that proteinases contribute to the generation of protein A fragments. We found that protein A fragments larger than about 6000 Da can be measured by enzyme linked immunosorbent assay, and that they can be more difficult to clear than whole protein A by cation-exchange chromatography.

Vanadium bromoperoxidase from Delisea pulchra: enzyme-catalyzed formation of bromofuranone and attendant disruption of quorum sensing.

Vanadium bromoperoxidase was isolated and cloned from the marine red alga Delisea pulchra. This enzyme catalyzes the bromolactonization of 4-pentynoic acid forming 5E-bromo-methylidenetetrahydro-2-furanone, a compound which is shown herein to inhibit quorum sensing in the engineered reporter strain, Agrobacterium tumefaciens NTL4.

The role of vanadium bromoperoxidase in the biosynthesis of halogenated marine natural products.

Halogenated natural products are frequently reported metabolites in marine seaweeds. These compounds span a range from halogenated indoles, terpenes, acetogenins, phenols, etc., to volatile halogenated hydrocarbons that are produced on a very large scale. In many cases these halogenated marine metabolites possess biological activities of pharmacological interest. Given the abundance of halogenated marine natural products found in marine organisms and their potentially important biological activities, the biogenesis of these compounds has intrigued marine natural product chemists for decades. Over a quarter of a century ago, a possible role for haloperoxidase enzymes was first suggested in the biogenesis of certain halogenated marine natural products, although this was long before haloperoxidases were discovered in marine organisms. Since that time, FeHeme- and Vanadium-haloperoxidases (V-HPO) have been discovered in many marine organisms. The structure and catalytic activity of vanadium haloperoxidases is reviewed herein, including the importance of V-HPO-catalyzed bromination and cyclization of terpene substrates.

Vanadium bromoperoxidase-catalyzed biosynthesis of halogenated marine natural products.

Marine red algae (Rhodophyta) are a rich source of bioactive halogenated natural products. The biogenesis of the cyclic halogenated terpene marine natural products, in particular, has attracted sustained interest in part because terpenes are the biogenic precursors of many bioactive metabolites. The first enzymatic asymmetric bromination and cyclization of a terpene, producing marine natural products isolated from red algae, is reported. Vanadium bromoperoxidase (V-BrPO) isolated from marine red algae (species of Laurencia, Plocamium, Corallina) catalyzes the bromination of the sesquiterpene (E)-(+)-nerolidol producing alpha-, beta-, and gamma-snyderol and (+)-3beta-bromo-8-epicaparrapi oxide. alpha-Snyderol, beta-snyderol, and (+)-3beta-bromo-8-epicaparrapi oxide have been isolated from Laurencia obtusa, and each have also been isolated from other species of marine red algae. gamma-Snyderol is a proposed intermediate in other bicyclo natural products. Single diastereomers of beta-snyderol, gamma-snyderol, and mixed diastereomers of (+)-3beta-bromo-8-epicaparrapi oxide (de = 20-25%) are produced in the enzyme reaction, whereas two diastereomers of these compounds are formed in the synthesis with 2,4,4,6-tetrabromocyclohexa-2,5-dienone (TBCO). V-BrPO likely functions by catalyzing the two-electron oxidation of bromide ion by hydrogen peroxide producing a bromonium ion or equivalent in the active site that brominates one face of the terminal olefin of nerolidol. These results establish V-BrPO's role in the biosynthesis of brominated cyclic sesquiterpene structures from marine red algae for the first time.

Structure and membrane affinity of a suite of amphiphilic siderophores produced by a marine bacterium.

Iron concentrations in the ocean are low enough to limit the growth of marine microorganisms, which raises questions about the molecular mechanisms these organisms use to acquire iron. Marine bacteria have been shown to produce siderophores to facilitate iron(III) uptake. We describe the structures of a suite of amphiphilic siderophores, named the amphibactins, which are produced by a nearshore isolate, gamma Proteobacterium, Vibrio sp. R-10. Each amphibactin has the same Tris-hydroxamate-containing peptidic headgroup composed of three ornithine residues and one serine residue but differs in the acyl appendage, which ranges from C-14 to C-18 and varies in the degree of saturation and hydroxylation. Although amphiphilic siderophores are relatively rare, cell-associated amphiphilic siderophores are even less common. We find that the amphibactins are cell-associated siderophores. As a result of the variation in the nature of the fatty acid appendage and the cellular location of the amphibactins, the membrane partitioning of these siderophores was investigated. The physiological mixture of amphibactins had a range of membrane affinities (3.8 x 10(3) to 8.3 x 10(2) M(-1)) that are larger overall than other amphiphilic siderophores, likely accounting for their cell association. This cell association is likely an important defense against siderophore diffusion in the oceanic environment. The phylogenetic affiliation of Vibrio sp. R-10 is discussed, as well as the observed predominance of amphiphilic siderophores produced by marine bacteria in contrast to those produced by terrestrial bacteria.

Vanadium haloperoxidase-catalyzed bromination and cyclization of terpenes.

Marine red algae (Rhodophyta) are a rich source of bioactive halogenated natural products, including cyclic terpenes. The biogenesis of certain cyclic halogenated marine natural products is thought to involve marine haloperoxidase enzymes. Evidence is presented that vanadium bromoperoxidase (V-BrPO) isolated and cloned from marine red algae that produce halogenated compounds (e.g., Plocamium cartilagineum, Laurencia pacifica, Corallina officinalis) can catalyze the bromination and cyclization of terpenes and terpene analogues. The V-BrPO-catalyzed reaction with the monoterpene nerol in the presence of bromide ion and hydrogen peroxide produces a monobromo eight-membered cyclic ether similar to laurencin, a brominated C15 acetogenin, from Laurencia glandulifera, along with noncyclic bromohydrin, epoxide, and dibromoproducts; however, reaction of aqueous bromine with nerol produced only noncyclic bromohydrin, epoxide, and dibromoproducts. The V-BrPO-catalyzed reaction with geraniol in the presence of bromide ion and hydrogen peroxide produces two singly brominated six-membered cyclic products, analogous to the ring structures of alpha and beta snyderols, brominated sesquiterpenes from Laurencia, spp., along with noncyclic bromohydrin, epoxide, and dibromoproducts; again, reaction of geraniol with aqueous bromine produces only noncyclic bromohydrin, epoxide, and dibromoproducts. Thus, V-BrPO can direct the electrophilic bromination and cyclization of terpenes.