Integrated solution to purification challenges in the manufacture of a soluble recombinant protein in E. coli.
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 at large scale by expression in E. coli. Significant difficulty with clearance of host cell proteins (HCPs) was experienced in the original manufacturing process despite a lengthy downstream purification train. Analysis of purified protein solutions and intermediate process samples led to identification of several major HCPs co-purifying with the product and a bacterial protease potentially causing a specific truncation of ApoA-1M found in the final product. Deletion of these genes from the original host strain succeeded in substantially reducing the levels of HCPs and the truncated species without adversely affecting the overall fermentation productivity, contributing to a much more efficient and robust new manufacturing process.
Pfizer Global Biologics, Chesterfield, Missouri 63017, USA.
Maire H
MH
Caparon
Kevin J
KJ
Rust
Alan K
AK
Hunter
Joseph K
JK
McLaughlin
Kristen E
KE
Thomas
John T
JT
Herberg
Robert E
RE
Shell
Paul B
PB
Lanter
Bruce F
BF
Bishop
Robert L
RL
Dufield
Xing
X
Wang
Sa V
SV
Ho
D000595:Amino Acid Sequence; D016632:Apolipoprotein A-I / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000302:isolation & purification; D017353:Gene Deletion; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D012995:Solubility
D016632:Apolipoprotein A-I; D029968:Escherichia coli Proteins; D011994:Recombinant Proteins; C080631:apolipoprotein A-I Milano