Proteins recombinant, human Interleukin

Hora et al. [3.19] described the complexity of protein stabilization by the example of recombinant, human Interleukin-2 (rhIL-2). Formulations with amino acids and mannitol/ sucrose are sensitive to mechanical stress e. g. by pumping. Hydroxypropyl-beta-cyclodextrin (HPcD) provides stability, but increases the sensitivity to oxygen. Polysor-bate 80 forms a mechanically stable product, but results in oxidation. In both cases contamination in the HPcD or traces of H202 in the Polysorbate may have been the starter for the oxidation. Brewster [3.20] reports, that HPcD stabilizes interleukin without forming aggregations and this results in 100 % biopotency. 

A protein whose degradation pattern is even more difficult to explain is recombinant human interleukin 2 (rhIL-2), a lymphokine produced by T-cells and being developed as an immunomodulator. This protein, which consists... 

Recombinant human interleukin-4 (rhIL-4) is a monomeric protein with a molecular weight of 15,400 Da and pi of 9.2, with three intrachain disulfide bonds. It is a cytokine that has been investigated for cancer therapy. CZE of rhIL-4 mixtures prepared by in vitro degradation has been performed in uncoated capillaries with 50 m M 1,3-diaminopropane and phosphate buffers with pH ranging from 4.5 to 8.O.40 The resolution of degradation products by CZE appeared to be superior to HPLC. 

Brent Pollock (Biomira Inc.) presented a talk on the rational development of a second-generation production process for recombinant human interleukin-2 that incorporates recent advances in cell expression systems, fermentation optimization, protein extraction, refolding, and purification. A key concept of this talk was to know thy protein. The driving forces for process changes were regulatory concerns/robustness, economics, the ability to use standard equipment, and the elimination of bottlenecks in the process. 

Recombinant human interleukin-2 (rIL-2, previously known as T-cell growth factor) was expressed in E. coli (7). During the purification process of rIL-2 by reversed-phase HPLC, another higher molecular weight form of this protein was also isolated, known as HMW rIL-2 (8). The purification process of HMW rIL-2 involved two chromatography steps and utilized a Bakerbond Carboxy-Sulfon (CS) column. Approximately 2 nmol each of rIL-2 and HMW rIL-2 were immobilized using ProSpin cartridges for C-terminal sequence analysis. 

USA oprelvekin protein Neumega Recombinant human interleukin-11 including residues 2-178, human clone pXM/IL-11, lacking original N-terminal Pro, expressed in E. coli Thrombo- cytopenia... 

Park, Y. and H. Cheong, Expression and production of recombinant human interleukin-2 in potato plants. Protein Expr Purif, 2002 25(1) 160-165. 

A15. Andus, T., Geiger, T., Hirano, T., Kishimoto, T., Tran Thi, T. A., Decker, K., and Heinrich, P. C. Regulation of synthesis and secretion of major rat acute-phase proteins by recombinant human interleukin-6 (BSF-2/IL-6) in hepatocyte primary cultures. Eur. J. Biochem. 173, 287-293 (1988). 

Recombinant human interleukin-11 oprelvekin (neumega) is a bacteiially derived, 19 kDa polypeptide that lacks the amino terminal Pro residue and is not glycosylated. The recombinant protein has a 7-hour after subcutaneous injection. In normal subjects, daily administration of oprelvekin leads to a thrombopoietic response in 5-9 days. 

Helqvist, S., Polla, B. S., Johannesen, J., and Nerup, J. (1991). Heat shock protein induction in rat pancreatic islets by recombinant human interleukin 1 /3. Diabetologia 34,150-156. 

Examples of the early application of recombinant DNA technology in medicine are the development of recombinant human growth hormone human insulin human interferons, thought to have anticancer activity in addition to antiviral activity interleukins (regulatory proteins from lymphocytes that are believed to be important in the treatment of immunodeficiency diseases and cancer) tumor necrosis factor epidermal and bone marrow progenitor cell growth factors and the production of vaccines (Table 12.1). 

A. General description Denileukin dif-titox is a recombinant, DNA-derived, interleukin-2 receptor specific ligand, cytotoxic fusion protein consisting of diphtheria toxin fragments A and B fused to interleukin-2. It is produced by expression of a recombinant fusion protein in Escherichia coli that contains nucleotide sequences for human interleukin-2, and sequences for the enzymatically active fragment A of diphtheria toxin and the membrane-translocating portion of diph-... 

IL-2, rDNA/Seragen [BIO] A-L-Methionyl-387-L-histidine-388-L-alanuie-l-388-toxin (Corynebacterium diphtheriae strain Cl) (388-2 ) protein with 2-133-interleukin 2 (human clone pTIL2-21a) [CAS] Interleukin-2 Fusion Protein [SY] Interleukin-2/diphtheria toxin fusion protein, recombinant [SY]... 

Denmark interleukin-2 protein Proleukin Macrolin Recombinant human IL-2 with N-terminal Ala and Cys 125 mutated to Ser, expressed in E. coli Anti-cancer... 

In the next contribution we learn about hands-on experience and recent improvements with different production systems for biopharmaceuticals at Bayer Health-Care. As previously also published in Nature by Heiner Apeler, Head of Expression, an E. coli host/vector system was originally developed for the efficient production of an interleukin-4 variant, but afterwards it was optimized for the expression of other proteins and even Fab fragments. Process development and optimization of the yeast secretory Saccharomyces cerevisiae for expression of a protease inhibitor will also be presented. The focus, however, is on the use of a recently developed mammalian HKBll (hybrid clone of human kidney and B cells) expression system for recombinant human glycoprotein biopharmaceuticals. HKBll is a favorable cell host for the production of human proteins, because it dehvers biopharmaceuticals that are structurally identical to the natural product. The host/vector system supports the production of gram quantities of proteins in a large-scale transient transfection format as well as the development of stable cell fines. These systems together... 

S., and Gauldie, J. Limited effects of recombinant human and murine interleukin-1 and tumour necrosis factor on production of acute phase proteins by cultured rat hepatocytes. Biochem. Inl. 14, 553-560 (1987). 

---