title: Process scale separation of an anti-CD22 immunotoxin charge variant.
abstract: We describe the analytical characterization and process scale separation of a deamidated variant of an immunotoxin. The different charge variants of the immunotoxin were separated using analytical ion-exchange HPLC. These charge variants were analyzed by peptide mapping and LC-MS/MS to identify the site of modification, which was determined to reside in the toxin portion of the molecule. Using a cell-based bioassay it was also determined that deamidation led to reduced biological activity, requiring it be controlled during manufacturing. This was accomplished using process scale anion-exchange chromatography. The process was capable of reducing the deamidated form to a level low enough for the resulting product to maintain acceptable biological activity. Keys to the successful control of this impurity at process scale were a good understanding of structure-function relationship and the availability of an analytical HPLC assay to provide a surrogate for the cell-based bioassay.
authors.affiliation: MedImmune, One MedImmune Way, Gaithersburg, MD 20878, USA.
authors.forename: Thomas
authors.initials: T
authors.lastname: Linke
authors.forename: Jinhua
authors.initials: J
authors.lastname: Feng
authors.forename: Kathy
authors.initials: K
authors.lastname: Yu
authors.forename: Hyun Jun
authors.initials: HJ
authors.lastname: Kim
authors.forename: Ziping
authors.initials: Z
authors.lastname: Wei
authors.forename: Yang
authors.initials: Y
authors.lastname: Wang
authors.forename: William K
authors.initials: WK
authors.lastname: Wang
authors.forename: Alan K
authors.initials: AK
authors.lastname: Hunter
mesh_terms: D036002:ADP Ribose Transferases / Q000737:chemistry; D000577:Amides / Q000737:chemistry; D001427:Bacterial Toxins / Q000737:chemistry* / Q000302:isolation & purification*; D045744:Cell Line, Tumor; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods*; D005098:Exotoxins / Q000737:chemistry* / Q000302:isolation & purification*; D006801:Humans; D000922:Immunotoxins / Q000737:chemistry* / Q000302:isolation & purification*; D010449:Peptide Mapping / Q000379:methods*; D053719:Tandem Mass Spectrometry; D037521:Virulence Factors / Q000737:chemistry; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D000577:Amides; D001427:Bacterial Toxins; D005098:Exotoxins; D000922:Immunotoxins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C541974:immunotoxin HA22