Conjugation of PolyPEG to interferon alpha extends serum half-life while maintaining low viscosity of the conjugate.

The covalent attachment of poly(ethylene glycol) (PEG) to therapeutic proteins is a commonly used approach for extending in vivo half-lives. A potential limitation of this PEGylation strategy is the adverse effect of PEG on conjugate viscosity. Interferon-alpha (IFN) was conjugated via its N-terminal amino group by reductive amination to α-aldehyde functional comb-shaped PolyPEG polymers (50 and 70 kDa) and to linear PEG (30 kDa). In vitro potencies of the purified PEGylated IFN conjugates were measured by reporter gene assay using a HEK293P/ISRE-SEAP cell line. IFN levels were measured in rats following intravenous injection. Viscosities of various linear PEG and PolyPEG polymers along with the polymer-IFN conjugates were determined using a rotational rheometer with cone-and-plate geometry. In vitro potencies and half-lives of the PEGylated IFN conjugates were compared with those of the marketed branched PEG-IFN conjugate PEGASYS. Both PolyPEG-IFN conjugates retained a similar potency as that of the marketed comparator, whereas the linear PEG-IFN conjugate potency was greater. All conjugates showed extended half-lives compared to that of naked IFN, with the PolyPEG conjugates exhibiting the longest half-lives and the linear PEG conjugate, the shortest. Viscosity analysis showed that the linear PEG-IFN conjugate was over twice as viscous as both PolyPEG conjugates. Taken together, this work demonstrates the potential of PolyPEG conjugation to therapeutic proteins as a novel tool for optimizing pharmacokinetic profiles in a way that potentially allows administration of high-dose formulations because of lower conjugate viscosity.

Early events in TNFa-p55 receptor interations--experiments with TNF dimers.

The first essential step in TNF signal transduction is believed to be clustering of the membrane bound receptors around the trimeric TNF molecule. To check if one receptor binding site would be enough to trigger the signal, we tried to prepare several types of TNF dimer. For this purpose, two TNF analogs bearing different cysteine mutations at the inner subunit binding surfaces were designed, expressed in E. coli and prepared in pure form. By mixing equimolar quantities of these analogs under appropriate conditions, two different types of dimer were prepared. The first, Dim/S2, proved to be composed mainly of a disulfide-linked dimer, which was still capable of trapping the third subunit of either of the precursor analogs, thus showing relatively high residual cytotoxicity. To avoid trimeric structures, Dim/S2 was further transformed into Dim/Iaa2 by alkylation of -SH groups of the newly introduced cysteines, allowing binding of only two TNF subunits through native contact surfaces. These dimers showed substantially reduced cytotoxicity on the L929 cell line. In addition, it appears that Dim/Iaa2 is able to competitively inhibit cytotoxicity of native TNF, as assessed on the L-M cell line.

Histidines in affinity tags and surface clusters for immobilized metal-ion affinity chromatography of trimeric tumor necrosis factor alpha.

In order to achieve efficient IMAC (immobilized metal-ion affinity chromatography) purification of tumor necrosis factor alpha (TNF-alpha) and its analogs by a common chromatographic procedure, we tested four histidine-rich affinity tags attached to the N-termini of the trimeric TNF-alpha molecule. Using low cultivation temperature and appropriate protease deficient E. coli strains, it was possible to obtain intact, full-length proteins with NHis2Xa and HisArg tags, which could be purified to over 95% purity in a single step. However, in comparison to model proteins bearing a surface histidine cluster, accumulation of the histidine-tagged proteins in E. coli was significantly reduced, even in protease deficient strains. In addition, the histidine tagged TNF-alpha proteins never displayed good chromatographic behavior, which was otherwise easily achieved with model proteins. Although the most popular hexa-histidine tag is generally recognized as very convenient for single step isolation of monomeric proteins, our results with trimeric TNF-alpha indicate that oligomeric proteins may require further optimization of the tag, with respect to its length, composition, and location. Histidines, relatively rigidly inserted in the structure, as in our model proteins, display superior chromatographic characteristics vis a vis flexible tags with the same total number of histidines.