Purification of clinical-grade disulfide stabilized antibody fragment variable--Pseudomonas exotoxin conjugate (dsFv-PE38) expressed in Escherichia coli.
Immunotoxins are rationally designed cancer targeting and killing agents. Disulfide stabilized antibody Fv portion-toxin conjugates (dsFv-toxin) are third generation immunotoxins containing only the antibody fragment variable portions and a toxin fused to the V(H) or V(L). Pseudomonas exotoxin fragment (PE-38) is a commonly used toxin in immunotoxin clinical trials. dsFv-toxin purification was previously published, but the recovery was not satisfactory. This report describes the development of a cGMP production process of the dsFv-toxin that incorporated a novel purification method. The method has been successfully applied to the clinical manufacturing of two dsFv-PE38 immunotoxins, MR1-1 targeting EGFRvIII and HA22 targeting CD22. The two subunits, V(L) and V(H) PE-38 were expressed separately in Escherichia coli using recombinant technology. Following cell lysis, inclusion bodies were isolated from the biomass harvested from fermentation in animal source component-free media. The dsFv-toxin was formed after denaturation and refolding, and subsequently purified to homogeneity through ammonium sulfate precipitation, hydrophobic interaction and ion-exchange chromatography steps. It was shown, in a direct comparison experiment using MR1-1 as model protein, that the recovery from the new purification method was improved three times over that from previously published method. The improved recovery was also demonstrated during the clinical production of two dsFv-PE38 immunotoxins-MR1-1 and HA22.
Biopharmaceutical Development Program, Frederick National Laboratory for Cancer Research, SAIC-Frederick Inc., Frederick, MD 21702, USA.
Hua
H
Jiang
Yueqing
Y
Xie
Andrew
A
Burnette
John
J
Roach
Steven L
SL
Giardina
Toby T
TT
Hecht
Stephen P
SP
Creekmore
Gautam
G
Mitra
Jianwei
J
Zhu
D000906:Antibodies / Q000737:chemistry* / Q000302:isolation & purification*; D006152:Cyclic GMP / Q000378:metabolism; D004220:Disulfides / Q000737:chemistry*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D005098:Exotoxins / Q000096:biosynthesis* / Q000235:genetics; D011549:Pseudomonas / Q000737:chemistry*
D000906:Antibodies; D004220:Disulfides; D005098:Exotoxins; D006152:Cyclic GMP