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.

Site of pegylation and polyethylene glycol molecule size attenuate interferon-alpha antiviral and antiproliferative activities through the JAK/STAT signaling pathway.

Therapeutic pegylated interferon-alphas (IFN-alpha) are mixtures of positional isomers that have been monopegylated at specific sites on the core IFN-alpha molecule. The pegylation results in lower in vitro specific activity associated with the core IFN-alpha 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-alpha2b that were monopegylated using 5-30-kDa linear PEG molecules attached at 7 primary reactive amino acid residues: Cys(1), His(34), Lys(31), Lys(83), Lys(121), Lys(131), and Lys(134). The isomers were evaluated for STAT translocation and antiviral and antiproliferative activity. The site of pegylation strongly influenced activity relative to an IFN-alpha2b control. The highest residual activity was observed with the His(34) positional isomers, and the lowest was observed with the Cys(1) positional isomers. The Lys positional isomers demonstrated intermediate activity, with a general order of Lys(134) > Lys(83) approximately Lys(131) approximately Lys(121) > Lys(31). The progressive relationship between decreased activity and increased PEG size suggests that pegylation may interfere with interaction and binding of IFN-alpha to the IFNAR1-IFNAR2 heterodimeric receptor. The higher specific activity associated with the His(34) positional isomer suggests that this site may be favorable for pegylating IFN-alpha2b molecules.

Characterization of hemodynamic events following intravascular infusion of recombinant adenovirus reveals possible solutions for mitigating cardiovascular responses.

Intravascular administration of recombinant adenovirus (rAd) in cancer patients has been well tolerated. However, dose-limiting hemodynamic responses associated with suppression of cardiac output have been observed at doses of 7.5 x 10(13) particles. While analysis of hemodynamic responses induced by small-molecule pharmaceuticals is well established, little is known about the cardiovascular effects of rAd. Telemetric cardiovascular (CV) monitoring in mice was utilized to measure hemodynamic events following intravascular rAd administration. Electrocardiogram analysis revealed a block in the SA node 3-4 min postinfusion, resulting in secondary pacemaking initiated at the AV node. This was associated with acute bradycardia, reduced blood pressure, and hypothermia followed by gradual recovery. Adenovirus-primed murine sera with high neutralizing antibody (nAb) titers could inhibit CV responses, whereas human sera with equivalent nAb titers induced by natural infection were, surprisingly, not inhibitory. Interestingly, repeat dosing within 2-4 h of the primary injection resulted in desensitization, resembling tachyphylaxis, for subsequent CV responses. Last, depletion of Kupffer cells prior to rAd infusion precluded induction of CV responses. These inhibitory effects suggest that rAd interactions with certain cells of the reticular endothelial system are associated with induction of CV responses. Significantly, these studies may provide insight into management of acute adverse effects following rAd systemic delivery, enabling a broadening of therapeutic index.

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 (H(2)L(2)) and half (HL) human IgG molecules, respectively. 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 (H(2)L(2)) 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 H(2)L(2)/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 H(2)L(2)/HL variations as a result of changes during the process-development stage of a mAb development program.