Marcio Voloch

Empty Capsids in Column-Purified Recombinant Adenovirus Preparations

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.

Carboxyalkylated Histidine Is a pH-Dependent Product of Pegylation with SC-PEG

AbstractPurpose. Pegylation of therapeutic protein usually results in a mixture of monopegylated proteins with differing sites of modification. With rh-interferon-α2A pegylation, we have found that this heterogeneity includes two classes of pegylation site chemistry, the relative proportions of which can be adjusted by reaction pH. Methods. The effect of pegylation reaction pH on the relative proportion of three peaks produced was investigated. Products were purified and characterized by peptide mapping, chemical stability to neutral hydroxylamine, and biologic activity. Results. Reactions at basic pH levels produced a mixture of products pegylated at lysine residues as has been observed elsewhere. However, the dominant product of reactions at mildly acidic levels of pH showed distinct chemistry and higher cytopathic effect activity. The primary site of modification at this pH was His34. We developed a quantitative assay using sensitivity to neutral hydroxylamine to measure the proportion of urethane bonds involving carboxyalkylated histidines. This assay showed that histidine was pegylated preferentially at low pH levels with another protein, rh-Interleukin-10. Conclusions. Reaction pH can be used to select the preferred pegylation site chemistry.

Improvement of Monoclonal Antibody Production in Hybridoma Cells by Dimethyl Sulfoxide

Hybridoma cultures are routinely used as a source for monoclonal antibody (mAb) production necessary for preclinical evaluation. However, these cultures typically have low volumetric and specific productivities. In this article, we examined the use and the timing of addition of dimethyl sulfoxide (DMSO) as a medium additive to improve mAb production in our hybridoma clone 19 (c19) cultures. From shake flask studies, we defined the optimal DMSO concentration and time of addition for improved productivity. This timing coordinated with high cell viability and density. Hybridoma cultures treated with DMSO up to 0.3% (v/v) possessed cell densities and viabilities comparable to untreated control. We demonstrated that 0.2% (v/v) DMSO added to shake flask cultures at their maximal viable cell densities resulted in a 2‐fold increase in specific mAb production. This procedure was scaleable up to 20 L Cellbags (Wave Bioreactors) with similar titer improvement. Moreover, DMSO treatment did not affect the bioactivity or glycosylation profiles of the mAb.

Detection and quantification of the human IgG4 half-molecule, HL, from unpurified cell-culture supernatants.

The presence and absence of inter‐heavy‐chain disulphide linkages contribute to the existence of the tetrameric (H2L2) and half (HL) human IgG molecules, respectively [Schuurman, Perdok, Gorter and Aalberse (2001) Mol. Immunol. 38, 1–8]. Reduced effector response in the human IgG4 subclass presents an alternative therapeutic platform in a monoclonal‐antibody (mAb) development program. During the initial cell‐selection stage, titres of the recombinant human antibody present in crude cell‐culture supernatants are determined by ELISA, a technique requiring nanogram quantities of mAb. In the case of an IgG4 antibody, this material is represented mainly by the combination of the tetrameric (H2L2) and dimeric (HL) forms of the antibody. The determination of concentrations or ratios of tetramer and dimer usually requires at least one chromatographic purification step, and thus frequently this is evaluated later in the mAb development process when the number of potential clones has been reduced. In the present paper we describe a Western‐blot‐based method that detects and quantifies IgG4 half‐molecules, HL, from crude cell‐culture supernatants without purification so that H2L2/HL ratios can be included as a part of early clonal evaluation along with the screening of mAb titres. This method was demonstrated (1) to have a linear HL detection range of 0.5–10 ng, (2) to require microlitre volumes of culture and (3) to react specifically with human IgG4 produced from hybridoma and Chinese‐hamster ovary cell cultures. Moreover, this protocol is applicable to evaluate and monitor potential H2L2/HL variations as a result of changes during the process‐development stage of a mAb development program.

Development of a Serum-free Suspension Process for the Production of a Conditionally Replicating Adenovirus using A549 Cells

Conditionally replicating adenoviruses (CRAVs) are a group of recombinant human adenoviruses genetically engineered to replicate in selected tissues, such as tumors. These viruses could potentially offer significant medicinal benefits, since the restrictive replication of these viral vectors leads to the lysis of target cells without harm to the surrounding tissues. The in vitro propagation and amplification of the CRAV vectors often requires special host cells with deregulated growth control pathways. In order to develop an efficient cell culture process for the scaleable production of a CRAV vector, A549 cells, a human lung carcinoma cell line normally cultured in adherent culture, were adapted to suspension culture. CRAV production was demonstrated with the suspension-adapted A549 cells and a baseline production process was developed in shake flasks. The ability to scale-up virus production was confirmed in stirred tank bioreactors. Molecular characterization of the suspension-adapted A549 cells indicates no significant changes in cellular mechanisms related to adenovirus infection.