Detection of contaminating enzymatic activity in plant-derived recombinant biotechnology products.
Residual impurities in recombinantly produced protein biologics, such as host cell proteins (HCP), can potentially cause unwanted toxic or immunogenic responses in patients. Additionally, undetected impurities found in recombinant proteins used in cell culture may adversely impact basic research and biotechnology applications. Currently, the enzyme-linked immunosorbent assay (ELISA) is the standard for detection of residual HCP contamination in recombinantly produced biologics. Alternatively, two-dimensional liquid chromatography coupled to mass spectrometry is being developed as a tool for assessing this critical quality attribute. Both of these methods rely on the direct detection of HCPs and some previous knowledge of the contaminant. For contaminating enzymes, the mass level of the impurity may fall below the threshold of detection of these methods and underestimate the true impact. To address this point, here we demonstrate facile detection and characterization of contaminating phytase activity in rice-derived recombinant human serum albumin (rHSA) using a sensitive, label-free nuclear magnetic resonance (NMR) spectroscopy assay. We observed varying degrees of phytase contamination in biotechnology-grade rHSA from various manufacturers by monitoring the degradation of adenosine-5'-triphosphate and myo-inositol-1,2,3,4,5,6-hexakisphosphate by (31)P NMR. The observed lot-to-lot variability may result in irreproducible cell culture results and should be evaluated as a possible critical quality attribute in plant-derived biotherapeutics.
Institute for Bioscience and Biotechnology Research, National Institute of Standards and Technology and the University of Maryland , 9600 Gudelsky Drive, Rockville, Maryland 20850, United States.
Robert G
RG
Brinson
Gary G
GG
Giulian
Zvi
Z
Kelman
John P
JP
Marino
D010832:6-Phytase / Q000032:analysis* / Q000378:metabolism*; D001709:Biotechnology / Q000379:methods*; D004340:Drug Contamination; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D012275:Oryza / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012709:Serum Albumin / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D012709:Serum Albumin; D010832:6-Phytase