Introduction: The ability to quantify systemic concentrations of protein therapeutics is complicated by the presence of endogenous analyte, specific binding proteins, and nonspecific matrix components in biological matrices (Lee & Ma, 2007). Further complications can be introduced following pegylation whereby polyethylene glycol (PEG) impedes epitope recognition. Due to substantial interference from high affinity binding proteins and the inability to measure systemic drug concentrations under normal conditions, acid dissociation was implemented to facilitate the pharmacokinetic evaluation of clinical samples containing pegylated human growth hormone (PEG-hGH).

Methods: A sandwich electrochemiluminescent immunosorbent assay (ECLA) was employed using an anti-PEG capture, anti-hGH detection format, thereby eliminating cross-reactivity with endogenous compound. Samples were acid treated with glycine buffered hydrochloric acid (approximately pH 2.0) to dissociate PEG-hGH from serum resident growth hormone binding protein (GHBP; 3). After neutralization with a HEPES-based neutralizing buffer, samples were diluted in a casein-based assay buffer containing 0.2% I-block, 0.1% Tween-20, 2M NaCl, and 10% normal mouse serum to eliminate nonspecific matrix effects. Meso Scale Discovery (MSD) technology was employed to achieve sensitivity requirements.

Results: The drug detection assay was validated in the presence and absence of acid dissociation. Validation parameters included: intra- and inter-assay accuracy and precision, selectivity (>60 lots of normal and growth hormone deficient human serum), cross-reactivity/specificity (Genotropin, hemoglobin, lipid, and bilirubin), dilutional linearity and stability (DeSilva et al., 2003; Shah et al., 1992; Smolec et al., 2005 Viswanathan et al., 2007; Food and Drug Administration, 2001). A 10-fold molar excess of GHBP was found to decrease PEG-hGH detection by >90% while acid dissociation was shown to recover >80% of the analyte. PEG-hGH clinical pharmacokinetic samples were analyzed with and without acid treatment. Only 38% of the mid-dose samples were quantifiable without acid treatment while 92% were quantifiable after acid dissociation.

Discussion: The implementation of acid dissociation was found to substantially increase the number of quantifiable pharmacokinetic samples over the drug exposure time course and contributed significantly to the robustness of pharmacokinetic evaluation.