Detection of residual polyethylene glycol derivatives in pyridoxylated-hemoglobin-polyoxyethylene conjugate

Purified hemoglobin solutions have been shown to cause renal toxicity in animals. Safe use of hemoglobin based therapeutics in humans requires modification of the hemoglobin molecule to prevent this toxicity. Hemoglobin modification may be accomplished by crosslinking the dimers within the hemoglobin tetramer or by derivatization of the alpha and/or beta subunits such that their size and/or charge prevents filtration by the glomeruli. Pyridoxylated hemoglobin polyoxyethylene conjugate (PHP) consists of hemoglobin molecules modified with alpha-carboxymethyl, omega-carboxymethoxy polyoxyethylene (POE). We have developed a high performance liquid chromatography-based (HPLC) method which can quantitate residual POE at levels of 0.1 mg/ml or greater. The detection of POE at this level of sensitivity requires the use of an evaporative light scattering detector (ELSD). A differential refractometer may also be used for POE detection, however the limit of quantitation for this detector is approximately 10 fold greater than that observed for the evaporative light scattering detector, resulting in a reduction in sensitivity. The successful use of this method requires sample deproteination using trichloroacetic acid. The reliability of the method has been demonstrated by spike recovery, precision, and reproducibility studies in PHP and buffer solutions.