Quantitation of E. coli protein impurities in recombinant human interferon-gamma

• Published in: Applied biochemistry and biotechnology Vol. 36; no. 2; p. 137
• Main Authors: Chen, A B, Championsmith, A A, Blanchard, J, Gorrell, J, Niepelt, B A, Federici, M M, Formento, J, Sinicropi, D V
• Format: Journal Article
• Language: English
• Published: United States 01.08.1992
• Subjects: Antibodies, Monoclonal; Bacterial Proteins - analysis; Blotting, Western; Cross Reactions; Drug Contamination; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Escherichia coli - chemistry; Humans; Interferon-gamma - chemistry; Interferon-gamma - isolation & purification; Recombinant Proteins
• ISSN: 0273-2289
• DOI: 10.1007/BF02929693

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.