title: Quantitation of E. coli protein impurities in recombinant human interferon-gamma.
abstract: A multiple antigen ELISA for E. coli proteins (ECPs) that may be present in purified recombinant human interferon-gamma (rIFN-gamma) was developed. SDS-PAGE and Western blotting analyses showed that the assay antibodies reacted with a wide spectrum of ECPs in the standard and with ECPs in a production run. In spike recovery studies, rIFN-gamma at concentrations of 0.05 mg/mL and higher augmented the immunoreactivity of the ECPs in the standard curve (1.3-40.0 ng ECPs/mL) by approx 50%. To determine ECP content in purified rIFN-gamma, 0.2 mg/mL of rIFN-gamma was added to the standard curve diluent to compensate for enhanced immunoreactivity. The assay was precise (interassay precision of ECP controls < or = 4.1 %CV) and accurate with recoveries of 111-115% of expected for ECPs (15-40 ng/mL) spiked into purified rIFN-gamma (1 mg/mL). Linearity of dilution for ECPs spiked into rIFN-gamma was obtained (r = 0.999). Moreover, linearity of dilution was obtained for ECPs in "in-process" samples, demonstrating the required condition of antibody excess for this type of multiple antigen ELISA. ECPs were not detectable in several purified lots of rIFN-gamma. Therefore, these lots contained < 1.3 ppm ECPs.
authors.affiliation: Department of Medicinal and Analytical Chemistry, Genentech, Inc., South San Francisco, CA 94080.
authors.forename: A B
authors.initials: AB
authors.lastname: Chen
authors.forename: A A
authors.initials: AA
authors.lastname: Championsmith
authors.forename: J
authors.initials: J
authors.lastname: Blanchard
authors.forename: J
authors.initials: J
authors.lastname: Gorrell
authors.forename: B A
authors.initials: BA
authors.lastname: Niepelt
authors.forename: M M
authors.initials: MM
authors.lastname: Federici
authors.forename: J
authors.initials: J
authors.lastname: Formento
authors.forename: D V
authors.initials: DV
authors.lastname: Sinicropi
mesh_terms: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins / Q000032:analysis*; D015153:Blotting, Western; D003429:Cross Reactions; D004340:Drug Contamination; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000737:chemistry*; D006801:Humans; D007371:Interferon-gamma / Q000737:chemistry / Q000302:isolation & purification*; D011994:Recombinant Proteins
chemical_list: D000911:Antibodies, Monoclonal; D001426:Bacterial Proteins; D011994:Recombinant Proteins; D007371:Interferon-gamma