Cetuximab

The cetuximab-IRDye800 immunoconjugate was produced under cGMP conditions at the University of Alabama (UAB) Vector Production Facility before shipment to Stanford University Hospital Pharmacy, which is nearly identical to that found in the literature.^{47 (link)} In brief, cetuximab (ImClone LLC, Eli Lilly and Company) was provided as a 2 mg/mL solution and concentrated, and the pH was adjusted by buffer exchange to a 10 mg/mL solution in 50 mmol/L potassium phosphate, pH 8.5. IRDye 800CW NHS ester (LI-COR Biosciences, Lincoln, NE) underwent conjugation to cetuximab for 2 h at 20 °C in the dark, whereby an average molar ratio of 1:2.3 cetuximab:IRDye800 was attained. After column filtration to remove unconjugated dye and exchange buffer to phosphate-buffered saline (PBS), pH 7, final protein concentration adjusted to 2 mg/mL, the product was sterilized by filtration and placed into single-use vials. After conjugation, the agent was shipped to Stanford University Hospital Pharmacy. During shipping of the immunoconjugate, the temperature was stable at 4 °C. The immunoconjugate was stored at 4 °C until used upon its receipt. The same lot was used for the cohort of animals in the treatment group.

Conjugation of cetuximab-IRDye800CW was performed under cGMP conditions, as previously described.(22 ) Briefly, cetuximab® (ImClone LLC, Eli Lilly and Company, Branchburg, NJ) was concentrated and pH adjusted by buffer exchange to a 10mg/ml solution in 50mM potassium phosphate, pH 8.5. IRDye800CW NHS ester (LI–COR Biosciences, Lincoln NE) was conjugated to cetuximab for 2hr at 20°C in the dark, at a molar ratio of 2.3:1. After column filtration to remove unconjugated dye and exchange buffer to phosphate buffered saline (PBS), pH 7, the final protein concentration adjusted to 2mg/ml, the product was sterilized by filtration and placed into single use vials and stored at 4°C until used.

Conjugation of cetuximab to IRDye800 was performed under cGMP conditions as previously reported (Zinn et al, Mol Imaging Biol, In Press). Briefly, cetuximab® (ImClone LLC, subsidiary of Eli Lilly and Company, Branchburg, NJ) was concentrated and pH adjusted by buffer exchange to a 10mg/ml solution in 50mM potassium phosphate, pH 8.5. IRDye 800CW NHS ester (LI-COR) was conjugated to cetuximab for 2hrs at 20ºC in the dark, at a molar ratio of 2.3:1. After column filtration to remove unconjugated dye and exchange buffer to phosphate buffered saline (PBS), pH 7, the final protein concentration adjusted to 2mg/ml, the product was sterilized by filtration and placed into single use vials and stored at 4°C until used.