James E. Seely

Use of ion-exchange chromatography and hydrophobic interaction chromatography in the preparation and recovery of polyethylene glycol-linked proteins

Cation- and anion-exchange chromatography can be used to purify a polyethylene glycol-linked protein dimer (PEG dimer) made with M, 20 000 PEG bis-vinylsulfone, even when there are no net charge differences between the components that are being separated. The retention time on ion-exchange generally is inversely proportional to the PEG:protein ratio (on a mass basis). One of the biggest challenges in developing the process for making this PEG dimer was the quality of the PEG linker. Reversed-phase HPLC can be used to determine both size heterogeneity and the degree of end-group activation of Mr 20 000 PEG bis-vinylsulfone. In addition, we have found that hydrophobic interaction chromatography can be used make more size homogeneous preparations of Mr 20000 PEG bis-vinylsulfone, which significantly increased the recovery of the PEG dimer.

Design of PEGylated soluble tumor necrosis factor receptor type I (PEG sTNF-RI) for chronic inflammatory diseases

A recombinant C-terminal truncated form of the human soluble tumor necrosis factor receptor type I (sTNF-RI) was produced in E. coli. This soluble receptor contains the first 2.6 of the 4 domains of the intact sTNF-RI molecule. A monoPEGylated form of this molecule was produced using a 30 kD methoxyPEG aldehyde with approximately 85% selectivity for the N-terminal amino group. This molecule was shown to be less immunogenic in primates than the full length (4.0 domain) molecule or other versions of sTNF-RI which were either PEGylated at different sites or with different molecular weight PEGs. The 30 kD PEG also has a longer serum half-life to the molecule than lower molecular weight PEGs. This molecule markedly blunts the inflammatory response in a number of rheumatoid arthritis animal models. In addition, phase I/II and early phase II data in humans indicate that PEG sTNF-RI is non-immunogenic and that weekly dosing with this drug can reduce the number of tender and swollen joints in rheumatoid arthritis patients. PEG sTNF-RI has comparable American College of Rheumatology (ACR) efficacy scores as other anti-TNF molecules currently used to treat rheumatoid arthritic patients.

Combination benefit of PEGylated soluble tumor necrosis factor receptor type I (PEG sTNF-RI) and dexamethasone or indomethacin in adjuvant arthritic rats

Abstract.Objective: To determine the potential combination benefit receptor of treatment with PEGylated soluble tumor necrosis factor type I (PEG sTNF-RI) and dexamethasone (dex) or indomethacin (indo) in adjuvant arthritic rats.¶Subjects: 160 male Lewis Rats.¶Treatment: PEG sTNF-RI, dex, indo.¶Methods: Rats with adjuvant arthritis were given daily oral dex (0.025 or 0.006 mg/kg) or indo (0.5 or 0.25 mg/kg) day 9-14, alone or in combination with PEG sTNF-RI (sc on days 9, 11, and 13 of arthritis). Efficacy was monitored by volume measurement of ankle joints, final paw weights and histologic evaluation with particular emphasis on bone lesions.¶Results: Treatment with 1 mg/kg PEG sTNF-RI alone resulted in 27% inhibition of final paw weights, dex alone (0.025 mg/kg) gave 25% inhibition and the combination resulted in 58% inhibition. Histologic evaluation of ankle joints demonstrated 48% inhibition of bone resorption with PEG sTNF-RI alone, 55% inhibition with dex alone and the combination treatment inhibited bone resorption by 100%. Inactive doses of PEG sTNF-RI (0.3 mg/kg) and dex (0.006 mg/kg) when combined resulted in 39% inhibition of paw swelling (AUC) and 39% inhibition of bone resorption. Combination treatment with indomethacin resulted in slight additive effects on inflammation parameters but no additive effects on bone resorption.¶Conclusion: Combination therapy with PEG sTNF-RI and dexamethasone results in additive or synergistic effects depending on the dose. Combination therapy with indomethacin resulted in slight additive effects on paw swelling parameters, but no additive benefit on bone resorption. Data from these studies support the clinical investigation of the use of combination therapy of PEG sTNF-RI and dex or other corticosteroids in rheumatoid arthritis patients.

Commercial production of recombinant erythropoietins

For the production of recombinant products from genetically engineered cells, a number of cell hosts may be used, but they generally belong to one of five categories: plant, bacterial, yeast, insect, or mammalian. Depending on the type of product that is desired, the features of that product, the intended use of the product, and the preferred method of manufacture, an appropriate host cell can be selected. With the appropriate genetic engineering, the gene of interest such as erythropoietin (EPO) can be produced by a host cell. In the case of EPO production, the sequence of amino acids, as well as the amount of glycosylation, must be correct to achieve the desired efficacy in vivo. This chapter describes one method of EPO production using cells genetically engineered to secrete recombinant human erythropoietin (rHuEPO). In this method of production, mammalian cells, which are capable of producing glycosylation forms with the desired efficacy in humans, are typically selected as hosts. The host mammalian cells secrete the rHuEPO product into the medium environment in which they are cultured, making the remainder of production a matter of separating the rHuEPO product from the cells and other components in the cell culture broth. This chapter summarizes the generation of rHuEPO-producing cell lines, the production of rHuEPO, the separation of rHuEPO from components of the cell culture broth, and the packaging of the final rHuEPO drug product.