Clearance of porcine circovirus and porcine parvovirus from porcine-derived pepsin by low pH inactivation and cation exchange chromatography.

The contamination of oral rotavirus vaccines by porcine circovirus (PCV) raised questions about potential PCV contamination of other biological products when porcine trypsin or pepsin is used in production process. Several methods can be potentially implemented as a safety barrier when animal derived trypsin or pepsin is used. Removal of PCV is difficult by the commonly used viral filters with the pore size cutoff of approximately 20 nm because of the smaller size of PCV particles that are around 17 nm. It was speculated that operating the chromatography step at a pH higher than pepsin's low pI, but lower than pIs, of most viruses would allow the pepsin to flow through the resin and be recovered from the flow through pool whilst the viruses would be retained on the resin. In this study, we investigated low pH inactivation of viruses including PCV Type 1 (PCV1) and PCV1 removal by cation exchange chromatography (CEX) in the presence of pepsin. Both parvovirus and PCV1 could be effectively inactivated by low pH and PCV1 could be removed by POROS 50HS CEX. The POROS 50HS method presented in this article is helpful for designing other CEX methods for the same purpose and not much difference would be expected for similar product intermediates and same process parameters. While the effectiveness needs to be confirmed for specific applications, the results demonstrate that both low pH (pH 1.7) and CEX methods were successful in eliminating PCV1 and thus either can be considered as an effective virus barrier.

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.

Balancing Efficacy and Safety of an Anti-DLL4 Antibody through Pharmacokinetic Modulation.

PURPOSE: Although agents targeting Delta-like ligand 4 (DLL4) have shown great promise for angiogenesis-based cancer therapy, findings in recent studies have raised serious safety concerns. To further evaluate the potential for therapeutic targeting of the DLL4 pathway, we pursued a novel strategy to reduce toxicities related to DLL4 inhibition by modulating the pharmacokinetic (PK) properties of an anti-DLL4 antibody. EXPERIMENTAL DESIGN: The F(ab')2 fragment of anti-DLL4 antibody (anti-DLL4 F(ab')2) was generated and assessed in efficacy and toxicity studies. RESULTS: Anti-DLL4 F(ab')2 enables greater control over the extent and duration of DLL4 inhibition, such that intermittent dosing of anti-DLL4 F(ab')2 can maintain significant antitumor activity while markedly mitigating known toxicities associated with continuous pathway inhibition. CONCLUSIONS: PK modulation has potentially broad implications for development of antibody-based therapeutics. Our safety studies with anti-DLL4 F(ab')2 also provide new evidence reinforcing the notion that the DLL4 pathway is extremely sensitive to pharmacologic perturbation, further underscoring the importance of exercising caution to safely harness this potent pathway in humans.

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.

Monoclonal antibody purification using cationic polyelectrolytes: an alternative to column chromatography.

The potential of cationic polyelectrolytes to precipitate host cell and process related impurities was investigated, to replace one or more chromatography steps in monoclonal antibody purification. The impact of antibody isoelectric point, solution properties (pH and ionic strength), and polyelectrolyte properties (structure, molecular weight and pK(a)) on the degree of precipitation was studied. At neutral pH, increasing solution ionic strength impeded the ionic interaction between the polyelectrolyte and impurities, reducing impurity precipitation. Increasing polyelectrolyte molecular weight and pK(a) enabled precipitation of impurities at higher ionic strength. PoIy(arginine) was selected as the preferred polyelectrolyte in unconditioned cell culture fluid. PoIy(arginine) precipitation achieved consistent host cell protein clearance and antibody recovery for multiple antibodies across a wider range of polyelectrolyte concentrations. Poly(arginine) precipitation was evaluated as a flocculant and as a functional replacement for anion exchange chromatography in an antibody purification process. Upstream treatment of cell culture fluid with poly(arginine) resulted in flocculation of solids (cells and cell debris), and antibody recovery and impurity clearance (host cell proteins, DNA and insulin) comparable to the downstream anion exchange chromatography step.

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.

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 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.

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.