Characterization of highly purified, inactivated HIV-1 particles isolated by anion exchange chromatography.

This report characterizes inactivated, gp120 depleted, HIV-1 particles purified by an anion exchange chromatography production process. This antigen formulated with incomplete Freund's adjuvant constitutes Remune, which is being evaluated in a phase III clinical endpoint trial to determine the effect of this immune-based therapy on clinical progression of HIV-1 seropositive patients. Multiple production lots of the inactivated HIV-1 antigen strain HZ321, isolated by anion exchange chromatography, exhibit purity of > 95% by gel filtration. These findings are corroborated by thin section electron microscopy showing a homogenous field of intact particles. Analyses of the purified virus particles for protein, lipid, carbohydrate and RNA show structural retention of the envelope proteins, lipid bilayer and core components after large scale processing. The qualitative identification of at least 85% of total HIV-1 protein is determined by ELISA, Western blot, HPLC and amino acid sequencing analyses. Quantitative values are assigned to 50% of these proteins. The data confirm the presence of virally encoded proteins p6, p7, pI15, p17, p24, p32, pI39Gag, gp41, pp55Gag, p66/51, Vpr, Vif and Nef. Excellent consistency between production lots and equivalency to HIV-1 preparations purified by sucrose density gradient sedimentation has been established for protein and lipid composition, and overall purity. These findings further establish that non-viral encoded proteins and lipids are integral structural components of the intact virion and are not contaminants unique to a particular isolation method. The data confirm the presence of multicomponent antigens in the viral particles for stimulating a broad HIV-1 specific immune response. Finally, the work demonstrates that the two inactivation procedures (beta-propiolactone and gamma irradiation), which achieve efficient viral inactivation meeting US FDA guidelines, do not damage the protein antigens of the viral particles.

Optimization of a recombinant von Willebrand factor fragment as an antagonist of the platelet glycoprotein Ib receptor.

The binding of von Willebrand factor (vWF) to platelet glycoprotein (GP) Ib receptor is one of the initial events in thrombus formation. Previous studies have shown that RG12986, a reduced and alkylated recombinant fragment of vWF (Ser445-Val733), can inhibit binding of native vWF to GP Ib and offers potential as an anti-thrombotic agent. We have now evaluated a series of deletion mutants of RG12986 and found that reduced and alkylated rvWF508-704 is close to the minimal sequence with optimal RG12986-like activity (IC50 for inhibition of GP Ib-dependent platelet aggregation in the absence of modulators: 0.022 microM +/- 0.01, n = 3) and that it too binds directly to GP Ib. Under in vitro conditions, with no exogenous modulators present and in the absence of shear stress, oxidized rvWF508-704 (containing a disulfide bond between Cys508 and Cys659) is approximately 5-fold less active than reduced and alkylated rvWF508-704; the two fragments, however, display comparable activity in the presence of the modulator botrocetin. The smaller rvWF508-704 fragment offers distinct advantages over RG 12986. In particular, removal of non-active NH2 and COOH terminal sequences may reduce the risk of antigenicity and may contribute to rendering the molecule mostly monomeric in solution, as opposed to the monomer-dimer equilibrium previously described for RG12986.

Different culture methods lead to differences in glycosylation of a murine IgG monoclonal antibody.

A monoclonal IgG-1 was produced by culture of a murine hybridoma (3.8.6) by three different methods, namely culture in ascites, in serum-free media and in serum-supplemented media. IgG-1 was purified to homogeneity (as judged by SDS/PAGE under reducing conditions) from each medium by ion-exchange chromatography and h.p.l.c. Protein A chromatography. Oligosaccharides were released from each IgG-1 preparation by hydrazinolysis and radiolabelled by reduction with alkaline sodium borotritide, and 'profile' analysis of the radiolabelled oligosaccharide alditols was performed by a combination of paper electrophoresis and gel-filtration chromatography. This analysis indicated clear and reproducible differences in the glycosylation patterns of the three IgG-1 preparations. Sequential exoglycosidase analysis of individual oligosaccharides derived from each IgG-1 preparation was used to define these differences. Ascites-derived material differed from serum-free-culture-derived material only with respect to the content of sialic acid. IgG-1 derived from culture in serum-containing media had an intermediate sialic acid content and a lower incidence of outer-arm galactosylation than the other two preparations. These differences in glycosylation could not be induced in any IgG-1 preparation by incubating purified IgG-1 with ascites or culture medium. It is concluded that the glycosylation pattern of a secreted monoclonal IgG is dependent on the culture method employed to obtain it.

The recovery of highly purified biopharmaceuticals from perfusion cell culture bioreactors.

The impact of continuous perfusion cell culture technology on production of one and two chain human rtPA, as well as monoclonal IgG and IgM, will be reviewed. Perfusion as compared to batch systems improve the quality of the product in the conditioned media in terms of biological activity and structural integrity. This significantly increases downstream recovery and daily production output of final purified material. These findings are a consequence of the ability of perfusion technology to 1) maintain cells intact at high cell density; 2) effectively reduce serum content to approximate serum free conditions; and 3) minimize residency time of labile product within the 37 degrees C environment of the bioreactor.

A new mass-spectrometric C-terminal sequencing technique finds a similarity between gamma-interferon and alpha 2-interferon and identifies a proteolytically clipped gamma-interferon that retains full antiviral activity.

A novel mass-spectrometric technique is described that permits the identification of the C-terminal peptide of a protein. The technique involves the incorporation of 18O into all alpha-carboxy groups liberated during enzyme-catalysed partial hydrolysis of the protein, followed by mass spectrometry to identify as the C-terminal peptide the only peptide that did not incorporate any 18O. The technique has been used to identify the true C-terminal tryptic peptide of a bacterially produced gamma-interferon and to distinguish it from a peptide produced by anomalous tryptic cleavage. It was found that a closely similar sequence segment of bacterially produced alpha 2-interferon undergoes an analogous cleavage. The technique was also used to identify the C-terminus of a clipped gamma-interferon that retains full antiviral activity.

Reversible changes in nucleosome structure and histone H3 accessibility in transcriptionally active and inactive states of rDNA chromatin.

The sulfhydryl reagent iodoacetamidofluorescein (IAF) was used to probe the structure of chromatin subunits in transcribed and nontranscribed regions of Physarum rDNA. IAF labels histone H3 -SH groups in the elongated monomeric subunits (A particles) from the transcribed region, but it does not label H3 in the 11S monomers from the nontranscribed central spacer. All H3 reactivity is lost from rDNA chromatin in the inactive spherule stage of Physarum. Restriction cleavage of rDNA chromatin generates fragments from the transcription unit with reactive H3 -SH groups, whereas fragments containing nontranscribed spacer sequences are unreactive. The extended rDNA chromatin contains all four core histones and other prominent proteins. Electron microscopy shows that most of the extended subunits consist of two roughly spherical bodies connected by a 50 bp nucleoprotein bridge.

Incorporation of exogenous pyrene-labeled histone into Physarum chromatin: a system for studying changes in nucleosomes assembled in vivo.

Treatment of Physarum histone with iodoacetoxypyrene selectively derivatizes a single H3 cysteine with acetoxypyrene. Microplasmodia can incorporate this AP-H3 into nucleosomes. The distinction between blue monomeric pyrene fluorescence and green excimer pyrene fluorescence allows detection of changes in distance between the closely positioned H3 cysteines in nucleosomes. Fluorescence of nucleosomes labeled in vivo with AP-H3 is almost exclusively of the excimer form, indicating that H3 cysteines are within a few angstroms of each other in the nucleosome core. In histones recovered from these nucleosomes all detectable pyrene is covalently bound to H3. When Physarum is exposed sequentially to labeled followed by unlabeled histone, there is a rapid appearance of green excimer emission in nucleosomes after addition of labeled histone and no apparent switch from excimer to monomer fluorescence after several replications of the genome in the presence of unlabeled histone. These experiments provide evidence in favor of a model for conservative distribution of nucleosomal histones during chromatin replication.