Conjugation of Panitumumab-IRDye800CW was performed under cGMP conditions, as described previously [3 (link)]. Briefly, Panitumumab (Vectibix; Amgen, Thousand Oaks, California, United States) was concentrated, and pH adjusted by buffer exchange to a 10 mg/mL solution in 50 mmol/L potassium phosphate, pH 8.5. IRDye800CW (IRDye800CW-N-hydroxysuccinimide ester, LI-COR Biosciences, Lincoln, Nebraska, United States) was conjugated to Panitumumab for 2 hours at 20°C in the dark, at a molar ratio of 2.3:1. After filtration with desalting columns (Pierce Biotechnology, Rockford, Illinois, United States) to remove unconjugated dye and buffer exchange to PBS, pH 7, the final protein concentration was adjusted to 2 mg/ml. The product was sterilized by filtration and placed into single-use vials and stored at 4°C until used.
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Covalent Conjugation of Panitumumab with IRDye800CW
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Corresponding Organization : Massachusetts General Hospital
Other organizations : Vanderbilt University Medical Center, Intuitive Surgical (United States), Stanford University, University of Wisconsin–Madison, Massachusetts Eye and Ear Infirmary
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Variable analysis
independent variables
- Molar ratio of Panitumumab to IRDye800CW used for conjugation (2.3:1)
- Final protein concentration of conjugated Panitumumab-IRDye800CW (2 mg/mL)
- Panitumumab (Vectibix; Amgen, Thousand Oaks, California, United States)
- IRDye800CW (IRDye800CW-N-hydroxysuccinimide ester, LI-COR Biosciences, Lincoln, Nebraska, United States)
- Conjugation reaction performed for 2 hours at 20°C in the dark
- Buffer exchange to PBS, pH 7
- Filtration to remove unconjugated dye and sterilize the final product
- Storage of the final product at 4°C
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