Therapeutic pegylated interferon-␣s (IFN-␣) are mixtures of positional isomers that have been monopegylated at specific sites on the core IFN-␣ molecule. The pegylation results in lower in vitro specific activity associated with the core IFN-␣ molecule that is related to the site of pegylation and size of polyethylene glycol (PEG) attached. We prepared purified, homogeneous, positional pegylation isomers of IFN-␣2b that were monopegylated using 5-30-kDa linear PEG molecules attached at 7 primary reactive amino acid residues: Chronic hepatitis C is considered one of the major causes of chronic liver disease, cirrhosis, and hepatocellular carcinoma and is the most common reason for liver transplantation in the United States (1). It is estimated that there are 3 million chronically infected individuals in the United States and over 170 million worldwide (1). Treatment of hepatitis C has evolved from the use of interferon-␣ (IFN-␣), 1 either alone or in combination with ribavirin, to the newer pegylated interferons (PEGIFNs), which have provided a dramatic increase in virological response, especially in combination with ribavirin. Standard IFN-␣ therapy has a short (Ͻ12-h) half-life that requires subcutaneous injection three times weekly to maintain effective levels in the blood (2). The short half-life of IFN-␣ has led to the development of longer lasting preparations achieved by the attachment of a large polyethylene glycol (PEG) molecule directly to IFN-␣. Two different commercial preparations of PEG-IFN-␣ have been developed for clinical use, PEG-IFN-␣2b (PEG-INTRON®) and PEG-IFN-␣2a (Pegasys®); both have long half-lives (40 and 80 h, respectively) that permit once weekly administration (3). Both of these preparations have been demonstrated to be effective for the treatment of patients with hepatitis C (4), and clinical trial results have shown further that both of the pegylated molecules produce sustained viral response rates superior to those achieved with their respective standard IFN-␣s (5-7).Whereas pegylation has proven to be highly effective for slowing the clearance of biological molecules, including IFN-␣, and thus increasing serum half-life, it has been shown to also modify in vitro biological activity (8). For instance, we have reported that pegylation of IFN-␣2b with a 12-kDa linear PEG molecule results in a preparation that has a specific activity of 28% relative to IFN-␣2b; the loss in activity was not due to structural perturbation of the core IFN-␣2b core protein (9). Other groups have reported that pegylation of IFN-␣2a with a 40-kDa branched PEG molecule results in a preparation that contains from 1 to 7% relative specific activity compared with IFN-␣2a (10, 11). These two pegylated interferon-␣s (PEG-IFN␣s) differ substantially in their postpegylation constituent properties. PEG-IFN-␣2b has a 12-kDa linear PEG molecule attached using succinimidyl carbonate polyethylene glycol (SC-PEG) chemistry via a covalent urethane-like bond to the IFN␣2b protein (12). The pegylation linkage process results...

No abstract

Empty capsids from adenovirus, that is, virus particles lacking DNA, are well documented in the published literature. They can be separated from complete virus by CsCl density gradient centrifugation. Here we characterize the presence of empty capsids in recombinant adenovirus preparations purified by column chromatography. The initial purified recombinant adenovirus containing the p53 tumor suppressor gene was produced from 293 cells grown on microcarriers and purified by passage through DEAE-Fractogel and gel-filtration chromatography. Further sequential purification of the column-purified virus by CsCl and glycerol density gradient centrifugations yielded isolated complete virus and empty capsids. The empty capsids were essentially noninfectious and free of DNA. Analysis of empty capsids by SDS-PAGE or RP-HPLC showed the presence of only three major components: hexon, IIIa, and a 31K band. This last protein was identified as the precursor to protein VIII (pVIII) by mass spectrometric analysis. No pVIII was detected from the purified complete virus. Analysis by electron microscopy of the empty capsids showed particles with small defects. The amount of pVIII was used to determine the level of empty capsid contamination. First, the purified empty capsids were used to quantify the relation of pVIII to empty capsid particle concentration (as estimated by either light scattering or hexon content). They were then used as a standard to establish the empty capsid concentration of various recombinant adenovirus preparations. Preliminary research showed changes in empty capsid concentration with variations in the infection conditions. While virus purification on anion-exchange or gel-filtration chromatography has little effect on empty capsid contamination, other chromatographic steps can substantially reduce the final concentration of empty capsids in column-purified adenovirus preparations.

No abstract

Ground corn selectively adsorbs water to give anhydrous product from ethanol vapors containing between 1.6 and 50.9% water. An adiabatic corn adsorber system is characterized by formation of a combined wave front (the temperature and concentration waves elute from the column at the same time). Corn is stable under cyclic use when regeneration is carried out wlth air or N2 at 80 to 120 O C . The energy required to regenerate the adsorbent is less than 1500 Btu/gal (528 kJ/kg) of anhydrous product obtained from ethanol vapor containing 7.4% water. Anhydrous product also results when water-containing vapors of n-propyl, isopropyl, or teff-butyl alcohol are passed over corn. Corn and other polysaccharide materials have potential as low-cost adsorbents for energy efficient water removal from alcohols. 0 1984 American Chemical Society 0 1 96-43051841 1 123-0437$Ol.50lO