Abstract: Apolipoprotein A-1 Milano (ApoA-1M), the protein component of a high-density lipoprotein (HDL) mimic with promising potential for reduction of atherosclerotic plaque, is produced on a large scale by expression in E. coli. Significant difficulty with clearance of host cell proteins (HCPs) was experienced in the original manufacturing process, despite lengthy purification. Analysis of purified protein solutions and intermediate process samples led to the identification of several major HCPs, and a bacterial protease causing the production of a truncated species of ApoA-1M co-purifying with the product. Deletions in these genes from the original host strain succeeded in substantially reducing the levels of HCPs without adversely affecting overall fermentation productivity, contributing to a much more efficient and robust new manufacturing process.

Abstract: The present invention relates to recombinant human interleukin-3 (hIL-3) variant or mutant proteins (muteins). These hIL-3 muteins contain amino acid substitutions and may also have amino acid deletions at both the N- and C- termini. The invention also relates to pharmaceutical compositions containing the hIL-3 muteins and methods for using them. Additionally, the present invention relates to recombinant expression vectors comprising nucleotide sequences encoding the hIL-3 muteins, related microbial expression systems, and processes for making the hIL-3 muteins using the microbial expression systems. Included in the present invention are deletion mutants of hIL-3 in which from 1 to 14 amino acids have been deleted from the N-terminus, and from 1 to 15 amino acids 119 to 133 have been deleted from the C-terminus, and which also contain amino acid substitutions in the polypeptide.